Prenatal screening

ABSTRACT

The present invention concerns products and methods particularly useful for activating and analyzing non-dividing cell nuclei. The featured products include activating egg extracts, cytostatic factor (CSF) extracts, kits containing these extracts, and a microchamber microscope slide useful in analyzing nucleus activation.

RELATED APPLICATION

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 08/013,039 entitled “Prenatal Screening” the whole of which ishereby incorporated by reference herein (including the drawings).

FIELD ON THE INVENTION

[0002] This invention concerns products, methods and apparatus foranalysis of non-dividing mammalian cell nuclei, such as human fetal cellnuclei and mammalian sperm cell nuclei.

BACKGROUND OF THE INVENTION

[0003] Jackson, Seminars in Perinatology 15:49 (1991), describes variousprocedures for prenatal diagnosis, including procedures to diagnosediseases. These procedures involve analysis of the DNA present in earlyembryonic stages. Specifically, Jackson mentions the use of a polymerasechain reaction to amplify genes, and the possibility of testing oocytesby polar body assay. According to Jackson:

[0004] “There are other conceivable embryo biopsy approaches forprenatal diagnosis. The trophectoderm may be obtained at later,multicellular embryonic stages when more cells might be obtained andinduced to replicate in tissue culture . . . . Another approach to earlyprenatal diagnosis is the recovery of fetal cells in the maternalcirculation. This tantalizing possibility for a non-invasive method hasbeen pursued for several years by groups in both the United States andthe United Kingdom. Both groups originally sought placental immunologicmarkers for identification and recovery of these cells. Severaltrophoblast antibodies were developed, some of which appeared to haverelative specificity for the fetal cell. After sporadic reports ofsuccess, recent articles appear to indicate that these markers areinsufficiently specific and actually are attached to maternal cellsfrequently enough to make this approach unworkable to date.”

[0005] Bianchi et al., Proc. Natl. Acad. Sci. USA 87: 3279 (1990),describe isolating fetal nucleated erythrocytes in maternal blood usinga monoclonal antibody against the transferrin receptor. They state thatthey “were successful in detecting the Y chromosomal sequence in 75% ofmale-bearing pregnancies, demonstrating that it is possible to isolatefetal gene sequences from cells in maternal blood.”

[0006] According to Roberts, Science 18:378 (1991), two proceduresavailable for prenatal screening are chorionic villus sampling (CVS) andamniocentesis. Both these procedures have problems involving waitingtime and risk of miscarriage, “estimated at 1% to 2% for CVS and 0.5%for amniocentesis.” Supra. Roberts also points out a procedure foranalyzing nuclear DNA directly when cells are in interphase.

[0007] Lohka and Masui, Science 220:719 (1983), describe inducing theformation of a nuclear envelope in demembraned sperm of Xenopus laevisusing a cell-free preparation from the cytoplasm of activated eggs ofRana pipiens.

[0008] Leno and Laskey, J. Cell Biology 112:557, (1991), performedexperiments using erythrocytes from adult chickens. According to Leno:

[0009] “Coppock et al. (1989) [Supra] have reported that a pretreatmentwith trypsin was required for nuclear decondensation and DNA replicationof Xenopus erythrocyte nuclei in egg extract. Trypsin pretreatment wasnot required for nuclear decondensation and DNA replication in ourextracts.”

[0010] Gordon et al., Experimental Cell Research 157:409 (1985),describe “a system for the activation of human sperm using cell-freeextracts from Xenopus laevis eggs.” Similarly, an abstract, by Brown etal., J. Cell Biology 99:396a (1984), indicate that nuclear changes whichoccur during the early phases of fertilization can be stimulated byinjecting isolated sperm nuclei into heterologous recipient eggs, or byincubating frog sperm nuclei in the presence of cell-free extracts fromfrog eggs. They state that they found human sperm can be activated invitro using Xenopus laevis frog egg extract to stimulate the earlyevents of nuclear activation, including chromatin decondensation,nuclear enlargement and DNA synthesis.

SUMMARY OF THE INVENTION

[0011] The present invention concerns products and methods useful forcausing non-dividing nuclei to activate (e.g., go through one or moresteps of nuclear activation). The featured products and methods areparticularly useful for activating human fetal cell nuclei and mammaliansperm cell nuclei. “Activation” of a non-dividing cell nucleus refers toone or more of the following activities: nuclear swelling, nucleic acidreplication, and nuclear entrance into mitosis thereby producingmetaphase chromosomes (arrested metaphase chromosomes or replicatingchromosomes). Complete activation refers to activation wherein all ofthe activities occur.

[0012] Nucleic acids can be analyzed at the different stages ofactivation, brought about by the present invention, to obtain usefulinformation such as information about nucleic acid structure, sequences,number of copies of a nucleic acid sequence, and nuclear location of anucleic acid. Analysis of nucleic acids can be carried out usingtechniques known in the art such as in situ hybridization and karyotypeanalysis of metaphase chromosomes.

[0013] One particular advantage of the present invention is its use inprenatal diagnosis. Activation of fetal cell nuclei can be used tofacilitate prenatal diagnosis of various human conditions. Nuclei fromof all types of human fetal cells including blood cells (such as redcells, white cells and other circulating cells of the fetus), as well asother types of fetal cells such as cells found in the amniotic fluid, orcells derived from the placenta (such as trophoblasts or syncytialtrophoblasts), can be activated using the described products andmethods. Preferably, the fetal cells to be activated are recovered fromthe blood or tissue of a pregnant woman rather than directly from thefetus or placenta, thereby decreasing the likelihood of discomfort orharm to the fetus and/or mother by the diagnosis procedure.

[0014] “Activation activity” refers to the ability of an agent to bringabout nuclear activation. Examples of agents which bring about nuclearactivation include a non-activated cytostatic factor (CSF) extract andactivating egg extract. Enhancement of activation activity refers to anincrease in the activation activity which is brought about by an agentwhich causes nuclear activation. Examples of agents which enhancenuclear activation caused by an activating agent include CSF extract,purified components thereof, and proteases.

[0015] Activation activity can be measured using techniques known in theart. Such techniques include microscopic visualization of swollennuclei, incorporation of labelled nucleic acid precursors into newlysynthesized nucleic acid, microscopic visualization of metaphasechromosomes, and in situ hybridization.

[0016] The featured methods include pretreating a non-dividing humannucleus to enhance its ability to activate, bringing about complete orpartial nuclear activation, and both bringing about and analyzing suchnuclear activation on a microchamber microscope slide. Other usefulmethods disclosed include preparing products such as an activating eggextract, a CSF extract, and a modified CSF extract; the Use of aprotease pretreatment step in the activation of sperm; an activationassay; a retroviral integration assay; and a procedure for cloning wholeanimals using activated nuclei.

[0017] The featured products including activating egg extract, CSFextract, kits containing these extracts, and a microchamber microscopeslide useful in analyzing nuclear activation, are also claimed as partof the present invention.

[0018] The nucleus of a non-dividing fetal cell or a sperm cell isnormally small, has condensed chromatin, and does not replicate ordivide. Specific nucleic acid sequences in the nucleus of these cellscan be stained by fluorescent in situ hybridization methods if thetarget nucleic acid sequence is accessible to the probe. However, thesmall size of the nucleus can affect the accessibility of particularnucleic acid sequences and the amount of information obtained fromsuccessful hybridization. Moreover, hybridization signals successfullyobtained are limited in spacial resolution by the size of the nucleus.As a result, obtaining a reliable fluorescent signal can be difficultand the information obtained by fluorescent staining generally indicatesonly the presence or absence of accessible specific sequences, andpossibly the number of such sequences per nucleus.

[0019] In the featured methods, the present invention brings about oneor more stages of nuclear activation: nuclear swelling, chromatindecondensation, DNA replication, and formation of metaphase chromosomes.Genetic information can be obtained from each of these stages, which arecharacterized by changes in nuclear structure and function. Usefulinformation obtained from these stages of activation includefacilitating the visualization of a particular chromosomal region usinga probe by increasing the spacial resolution during swelling therebyincreasing accessibility of the chromosomal region to the probe;detecting the number of a particular type of chromosome initiallypresent by determining the increased number of the particular chromosomebrought about by replication; and visualizing chromosomal morphology bystaining metaphase chromosomes, including the presence of one or moresequences at specific locations within chromosomes.

[0020] Thus, in the first aspect, the invention features a method forcausing a nucleus from a human fetal cell to activate. Activation isbrought about by contacting a pretreated or, preferably, a furtherpretreated nucleus, with activating egg extract.

[0021] The present invention can be used to study fetal cell nuclei acidisolated by different procedures. For example, fetal cells can beobtained from circulating maternal blood, or by techniques such asamniocentesis or chorionic biopsy. Preferably, the fetal cell isobtained in a non-invasive manner (e.g., without disturbing the womb).Fetal cells such as erythrocytes and leukocytes cross the placenta andcirculate transiently in maternal blood. Furthermore, trophoblasts whichform the outermost placenta layer can pinch off and circulate inmaternal blood. Trophoblasts typically end up trapped in the maternallung capillary network.

[0022] Nuclear isolation and pretreatment is preferably carried outusing mild conditions. Mild conditions are those which allows fornuclear isolation and pretreatment while causing the minimal amount ofprotein and nucleic acid damage. Using mild conditions helps maintainthe integrity of the nucleic acid thereby decreasing artifacts duringsubsequent staining, and prevents premature protease activation therebyallowing subsequent protease treatment to occur under controlledconditions chosen to optimize such treatment.

[0023] Preferably, nuclear isolation and pretreatment to release anucleus from its surrounding cytoskeleton thereby forming a pretreatednucleus is carried out in two steps; (1) membrane permeabilization, and(2) separation or alteration (e.g., denaturation or degradation) ofcytoskeletal proteins and nuclear matrix proteins. These steps may becarried out simultaneously or separately. Formation of a pretreatednucleus is preferably carried out under conditions minimizing nucleicacid damage and damage to histones.

[0024] Membrane permeabilization, opens up the membrane therebyfacilitating subsequent nuclear treatment. Different techniques may beused for membrane permeabilization including hypotonic shock, shearingand detergent. Preferably a non-ionic detergent is used to permeabilizethe plasma and nuclear membranes. More preferably, lysolecithin is usedas the non-ionic detergent.

[0025] Different procedures can be use to separate, denature, anddegrade the cytoskeletal proteins surrounding the nucleus and nuclearmatrix proteins within the nucleus. These procedures include the use ofa thiol reducing, agent to denature nuclear protein, using controlledsalt extraction to selectively remove cytoskeletal and nuclear matrixproteins, and using controlled poly-anionic treatment to facilitateseparation of negatively charged nucleic acid from the positivelycharged nuclear proteins. Separation conditions should be chosen toensure a minimal amount of damage to nucleic acids, histones, andnon-cytoskeletal proteins. Preferably, a protease is used under mildconditions to remove cytoskeletal proteins surrounding the nucleus. Morepreferably, trypsin is used as the protease. In the most preferredembodiment, pretreatment is achieved using trypsin and lysolecithin.

[0026] Activating egg extracts are used to bring about nuclearactivation. Activating egg extracts contain material, such asprecursors, protein(s), nuclear envelope vesicles and mRNA, whichsupport nuclear activation. An egg can be chemically, physically, orelectrically induced to produce material which brings about nuclearactivation. Eggs can be induced using a calcium ionophore as describedbelow. The induced egg continues in its cell cycle. It appears that whenan egg is at the point in the cell cycle just prior to the S-phase, theegg cytoplasm is most active in supporting activation. As the eggproceeds into and past the S-phase, it appears to produce materialinhibitory to activation.

[0027] Preferably activating egg extracts are prepared from Xenopuseggs. More preferably activating egg extract are prepared from eggshaving an elevated DNA synthesis activation activity. Activating eggextract prepared from Xenopus eggs induced for 10 minutes at 20° C.contain approximately 59% of the optimal DNA synthesis activationactivity of Xenopus eggs induced for 25 minutes at 20° C. At about 25-30minutes at 20° C. the Xenopus eggs are at highest (optimal), or peak,DNA synthesis activation activity. Xenopus eggs induced for 40 minutesat 20° C. appear to have a lower DNA synthesis activation activity thanthe peak activation activity. Thus, the present invention discloses theuse of induced eggs having an elevated DNA synthesis activation activityof 70% or greater of the peak activation activity.

[0028] Activating extracts prepared from Xenopus eggs induced for 10minutes or less at 20° C. produce a lower rate of DNA replication intreated nuclei. However, activating extract prepared from Xenopus eggsinduced for 10 minutes at 20° C. appear to produce equivalent or greaternuclear swelling in treated nuclei than extracts prepared from Xenopuseggs induced for more than 10 minutes at 20° C.

[0029] More preferably activating egg extract is prepared from a numberof eggs (e.g., 1,000 to 10,000), most or all of which have an elevatedor peak DNA synthesis activation activity. Obtaining a large number ofeggs having a peak or elevated DNA synthesis activation activity ispreferably achieved using hardened eggs which have been synchronouslyinduced. Hardened eggs are prepared by hardening the vitelline envelopesurrounding the egg (described in detail below). Hardened eggs are lesslikely to spontaneously activate than soft non-hardened eggs.

[0030] Thus, by using hardened eggs a large number of eggs can becollected and induced at the same time (synchronously induced). A givennumber of eggs synchronously induced should all be at or near the samepoint in their cell cycle at a given later time. A large number of eggshaving an elevated DNA synthesis activation activity can be obtained byinducing the eggs at one time, and preparing the activating egg extractfrom all the eggs at a second later time. Preferably the activating eggextract is stored frozen. Freezing the extract allows a large amount ofextract to be prepared at one time and used at different later times.

[0031] Various supplements to activating egg extract have been found toincrease the activation activity of the activating egg extract. Thesesupplements include cell cycle regulatory proteins, cell cycleinhibitors, cAMP (preferably, between 0.1 and 1.0 mM, most preferably at0.3 mM), and phosphodiesterase inhibitors (preferably caffeine, morepreferably caffeine at a concentration between 0.1 and 10.0 mM, mostpreferably caffeine at a concentration of 1 mM).

[0032] In another preferred embodiment activation occurs under nuclearnon-duplication conditions wherein the nucleus swells, replicates DNA,forms metaphase chromosomes and prepares to divide (i.e., entersmitosis), but segregation of sister chromatids is prevented byinhibiting spindle formation. The inhibition of spindle formationprevents the division of the cell nucleus and the resulting separationof metaphase chromosomes.

[0033] Thus, under non-duplication conditions metaphase chromosomes aredetectable for a longer time period and are provided in a “spreadpattern.” A “spread pattern” refers to the orientation of differentchromosomes with respect to each other. Drugs such as nocodazole,colchine, or colcemid can be used to inhibit spindle formation.Preferably nuclear non-duplication conditions is achieved by addingnocodazole to the activating egg extract. More preferably, nocodazole isin an amount which will not inhibit DNA replication (e.g., less than 5μg/ml).

[0034] In other preferred embodiments, prior to being treated with theactivating egg extract, the pretreated nuclei are further pretreated bycontact with a CSF extract, or a purified component of the CSF extractincluding a purified kinase or a purified phosphatase. By “purified” ismeant the component is more concentrated (e.g., has a higher specificacitivity) than when present in a CSF extract. The desired purifiedkinase or phosphatase can be obtained by purifying the enzymes from CSFfractions and assaying for activation activity. Further pretreatmentwith CSF is preferably carried out under conditions not resulting innucleus activation. Premature activation occurring under non-controlledconditions decreases the ability of CSF extracts to enhance activationbecause activation is occurring in CSF extract under non-optimizedconditions. Another disadvantage of premature activation is that itproduces a pool of nuclei activated at different times, which is moredifficult to examine than nuclei activated at the same time.

[0035] CSF extracts can be used to increase nuclear activation uponsubsequent contact with an activating agent. CSF extracts can beprepared from non-induced eggs (i.e., eggs arrested in meiotic metaphaseII or activated eggs that have been arrested in mitotic metaphase).These extracts contain factors which aid in nuclear activation, such asCSF and mitosis promoting factor (MPF). MPF may help bring aboutactivation and visualization of chromosome by stimulating chromosomecondensation and inhibiting spindle assembly.

[0036] A preferred source of CSF extracts is Xenopus eggs. Isolation ofCSF extract from Xenopus eggs is facilitated using “hardened eggs” whichdo not spontaneously induce. Preferably, the CSF extract is storedfrozen. Freezing the extract allows a large amount of extract to beprepared at one time and used at different later times.

[0037] CSF extract is preferably supplemented with reagents such asβ-glycerol phosphate, creatine phosphate, phosphocreatine kinase, andCa²⁺ in amounts which improves activation of nuclei in activatingextract, without causing the start of the cell cycle prior to contactwith activation egg extract. Preferably, the CSF extract contains Ca²⁺in an amount which leads to an increase in the level of histone kinaseor MPF activity without initiating the cell cycle. The use of Ca²⁺ tosupplement CSF extract is particular advantageous if the CSF extract isfrozen before use. The Ca²⁺ may be added before freezing or afterthawing.

[0038] Ca²⁺ is a cofactor for calmodulin activated protein kinases andmay increase CSF activity by increasing the level of phosphorylatedtopoisomerase II activity. Topoisomerase II is a scaffold protein whichaids in chromosome decondensation and condensation possibly by anchoringchromatin loop domains. Wood and Earnshaw, J. Cell Biology 111:2839(1990). Ca²⁺ also appears to increase the histone kinase level, which wehave used as one measure of MPF activity.

[0039] As would be appreciated by one skilled in the art, the optimalamount of Ca²⁺ added to a CSF extract varies depending upon the presenceof a Ca²⁺ chelator. The Ca²⁺ concentration is preferably equal to orgreater than 100 μM; more preferably the Ca²⁺ concentration is between100 μM and 400 μM. These preferred concentrations were determined usinga CSF extract supplemented with 1 mM ethylene glycol-bis(β-aminoethylether)N,N,N′N′-tetraacetic acid (EGTA).

[0040] In another preferred embodiment, nuclei are activated undernon-synthesis conditions which inhibit nucleic acid synthesis. As aresult, the nucleus swells with or without formation of a nuclearenvelope but does not replicate DNA or enter mitosis. The resultingincreased spacial resolution brought about by nuclear swellingfacilitates the use of nucleic acid probes by making regions of nucleicacid more accessible. Non-synthesis conditions, which neverthelesspermit nuclear swelling may be achieved by the addition of reagents suchas aphidicolin (e.g., 50-100 μg/ml), 6-dimethylaminopurine (e.g., at 5mM), leupeptin (e.g., at 5 μg/ml) dideoxycytidine triphosphate (e.g.,0.1 mM) or dideoxythymidine triphosphate (e.g., 0.1 mM) to an activatedegg extract, or to CSF extract which is then contacted with an activatedegg extract.

[0041] In another aspect, a non-dividing human nucleus is furtherpretreated for subsequent activation by contact with a purified proteinkinase or a purified phosphatase which is present in a CSF extract. Thepurified protein kinase or purified phosphatase is in a purer form(e.g., more concentrated or more active) than that found in a CSFextract.

[0042] In a third aspect, the invention features a method for activatinga non-dividing human nucleus by further pretreating the non-dividinghuman nucleus in CSF extract, prepared from hardened eggs, and thencontacting these further pretreated nuclei with an activating eggextract prepared from synchronously induced hardened eggs.

[0043] In preferred embodiments the CSF extract is frozen before use andthe activating egg extract is frozen before use.

[0044] In other preferred embodiments, pretreated nuclei undergo furtherpretreatment in CSF extract involving a warm-then-cold incubationregime. Both the warm and cold steps increase activation of nuclei uponsubsequent contact with activating extract. Preferably, incubation iscarried out at about 25° C. for at least 30 minutes followed byincubation at about 4° C. for at least 30 minutes. Less preferred, butstill an effective incubation, is a warm regime at about 25° C. for atleast 30 minutes.

[0045] In other preferred embodiments, thawed CSF extract issupplemented with Ca²⁺ in an amount which does not start the cell cyclebut improves nuclei activation. The Ca²⁺ should be in an amount whichleads to an increase in the level of histone kinase or MPF withoutinitiating the cell cycle.

[0046] In another aspect, methods are described for preparing anactivating egg extract, from hardened eggs, which can cause non-dividinghuman nuclei cells to activate. The activating egg extracts are preparedfrom hardened eggs which have been synchronously induced such that theactivating egg extract is prepared from eggs having an elevated DNAsynthesis activation activity. Preferably synchronous induction iscarried out using eukaryotic cells, more preferably amphibian, yeast,human, echinoderm, mollusc, or fish, or chicken cells are used; morepreferably Xenopus eggs are used; even more preferably Xenopus eggsinduced for more than 10 minutes are used; most preferably Xenopus eggsare induced for 25-30 minutes at 20° C.

[0047] In another aspect a method for inducing swelling in non-dividingnuclei is described. The method can be used to induce swelling in theabsence of an activating extract and in the absence of DNA synthesis. Inparticular, CSF extract is supplemented with a protein kinase inhibitorand/or an aqueous solution.

[0048] In another aspect, a method for chromosome formation without DNAreplication is described. The method involves using a CSF extractsupplemented with a cyclin such as cyclin-

90 in an amount sufficient to enhance nuclear envelope breakdown andnuclear chromosome formation. The cyclin is thought to act by raisingthe level of MPF activity in a CSF extract.

[0049] In another aspect, a method for activating a mammalian sperm cellnucleus is described. The method involves the steps of: (a) pretreatinga sperm cell, using a membrane permeabilizer, a protease, and a thiolreducing agent to form a pretreated sperm cell; and (b) activating thepretreated sperm cell. The method can be used to study sperm fromdifferent mammals. Such studies can be carried out, for example, todetermination whether the sperm contains a particular gene or nucleicacid sequence which can be passed on during fertilization.

[0050] In another aspect, activation assays are described. These assayscan be used to measure different stages of activation. A basic assaycomprises isolating a nucleus, pretreating the nucleus, furtherpretreating the nucleus, contacting the further pretreated nucleus withan activating egg extract and measuring activation activity. Measurementof activation activity can be carried out using standard techniques suchas incorporation of labelled nucleotides into newly synthesized nucleicacid and microscopic visualization of nuclear swelling and metaphasechromosome formation.

[0051] Other activation assays are performed by altering one or more ofthe steps of the basic assay. For instance, to assay for importantfactors in CSF extract, rather than using whole CSF extracts, fractionsof the extract can be used. These fractions are obtained using standardpurification techniques. Similarly, different activating egg extractfractions can be studied.

[0052] In a preferred embodiment, a sperm activation assay, particularlyuseful to study human male fertility, is described. Uses of the spermactivation assay include, determining the effect of handling sperm underdifferent condition thereby obtaining optimal handling condition forsubsequent in vitro fertilization, and testing the effect of possiblemale contraceptives on activation.

[0053] In other aspects viral integration assays involving the use of acell nucleus or a pseudonucleus are described. Viral integration into acell nucleus can be assayed as follows: pretreating a cell nucleus toseparate the nucleus from its surrounding cytoskeleton; activating thepretreated nucleus and incubating with a viral integration complexcontaining viral nucleic acid; and measuring integration of viralnucleic acid into nucleic acid of the cell nucleus. The viralintegration complex containing viral nucleic acid can be added atdifferent times during nuclear pretreatment and activation.

[0054] The viral integration assay using a pseudonucleus involves: a)constructing a pseudonucleus from a defined DNA template; b) replicatingthe pseudonucleus; and c) incubating the pseudonucleus in the presenceof a viral integration complex containing viral nucleic acid. Thisintegration complex can be added at any time during pseudonucleusformation or replication. A pseudonucleus can be constructed, forexample, by adding plasmid DNA to a CSF extract or an activatingextract. The plasmid forms chromatin in the CSF extract but does notreplicate until Ca²⁺ (1-4 mM) is added. Activation of the extractcontaining the pseudonucleus leads to nuclear envelope formation aroundthe chromatin template and causes the chromatin to replicate.

[0055] In another aspect, a product for further pretreatment of nucleiis described. The further pretreatment product comprises CSF withextract supplemented with Ca²⁺. Ca²⁺ is provided in an amount whichincreases nuclei activation upon subsequent contact with activatingextract. Preferably, the CSF extract is also supplemented withβ-glycerol phosphate, creatine phosphate, and phosphocreatine kinase,

[0056] In preferred embodiments the CSF extract is frozen; the Ca²⁺concentration is equal to or greater than 100 μM; more preferably theCa²⁺ concentration is between 100 μM and 400 μM. These preferredembodiments were determined using a CSF extract supplemented with 1 mMEGTA.

[0057] In another aspect, a product for causing nuclear to swelling isdescribed. The product contains CSF extract supplemented with a proteinkinase inhibitor and/or an aqueous solution.

[0058] In another aspect a product for causing chromosome formationwithout DNA replication is described. The product is made up of a CSFextract supplemented with a cyclin such as cyclin-

90 in an amount sufficient to bring about nuclear envelope breakdown andnuclear chromosome formation.

[0059] In another aspect, a product for causing a non-dividing nucleusto activate is described. The activating product comprises an activatingegg extract prepared from an egg(s) having an elevated DNA synthesisactivation activity.

[0060] In a preferred embodiment activating egg extract is prepared fromXenopus eggs synchronously induced for more than 10 minutes; preferablythe Xenopus eggs are induced for 25 to 30 minutes at about 20° C.

[0061] In other preferred embodiments, the activating egg extract ismodified by supplementation with cell cycle regulatory proteins, cellcycle inhibitors, cAMP (preferably, between 0.1 and 1.0 mM, mostpreferably at 0.3 mM), and phosphodiesterase inhibitors (preferablycaffeine, more preferably caffeine at a concentration between 0.1 and10.0 mm, most preferably caffeine at a concentration of 1 mM).

[0062] In another aspect, a kit is disclosed for activating anon-dividing nucleus. The kit is comprised of frozen activating eggextract prepared from an egg having an elevated DNA synthesis activationactivity and frozen CSF extract.

[0063] In a preferred embodiment the CSF extract contains Ca²⁺. In amost preferred embodiment the kit contains a microchamber microscopeslide.

[0064] In another aspect, the invention features a microchambermicroscope slide provided with an upper surface having a water-repellentmaterial of a known thickness defining a microchamber on the uppersurface. The microchamber is shaped to enhance flushing of themicrochamber, and connected by at least one channel to a well on theupper surface.

[0065] In preferred embodiments, the microchamber is teardrop-shaped orpear-shaped; preferably two wells are provided at opposite ends of themicrochamber connected by two separate channels to the microchamber; andthe microchamber has a defined volume preferably between 5 and 50 μl,more preferably between 10 and 20 μl when a coverslip is placed over it.Fluid can be introduced into the microchamber by placing fluid in onewell and allowing it to flow through the microchamber to the oppositewell. The fluid is then removed from the opposite well. Removal may beachieved by pipetting away the fluid or by capillary action by placementof a filter paper within the well.

[0066] In other preferred embodiments, the water-repellent material is atape or a coating on the upper surface of the slide, more preferably aTEFLON® coating, or a wax film (e.g., a PARAFILM®). In most preferredembodiments, the upper slide surface is treated to enhance cell growthcompared to an untreated slide, the slide is provided in a sterilecondition, and/or the slide is coated with an antibody able tospecifically bind to a human fetal cell.

[0067] The advantages of the present invention include, but are notlimited to, facilitating prenatal screening by optimizing conditions fornuclear activation, which causes the nucleus of a fetal cell to eitherswell, replicate nucleic acid, and/or form metaphase chromosomes.Important information regarding nucleic acid sequences or chromosomemorphology can be readily obtained from these various stages ofactivation, for example by using DNA probes or visualizing the producedmetaphase chromosomes. Because some fetal cells, such as trophoblasts,erythrocytes, and leukocytes can be obtained from a maternal source, anadvantage of the invention is a non-invasive procedure to detect thepresence of genetic defects in such cells.

[0068] Other features and advantages of the invention will be apparentfrom the following description of the preferred embodiments thereof, andfrom the claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0069] The drawings will first briefly be described.

[0070] Drawings

[0071]FIG. 1 shows the effect on DNA replication of activated nuclei, ofusing frozen/thawed activating egg extracts supplemented with caffeine.

[0072]FIG. 2 shows the effect on DNA replication of activated nuclei, ofusing CSF extract supplemented with 6-dimethylamino-purine (DMAP).

[0073]FIG. 3 shows the effect of various warm-then-cold protocols on DNAreplication in activated nuclei.

[0074]FIG. 4 is a top view of a microchamber microscope slide.

METHODS AND PRODUCTS

[0075] Methods for activating nuclei include those described by Coppocket al., Developmental Biology 131:102 (1989); Wangh, J. Cell Science93:1 (1989); Wood and Earnshaw, J. Cell Biology 111:2839 (1990); Lenoand Laskey, J. Cell Biology 112:557 (1991); Young, Biology ofReproduction 20:1001 (1979); Philpott et al., Cell 65:569 (1991); Shamuand Murray, J. Cell Biology 117:921 (1992); Adachi et al., Cell 64:137(1991); Newport and Spann, Cell 48:219 (1987); and Henry Harris, in CELLFUSION 40-50 (Harvard University Press 1970).

[0076] DiBerardino et al., Proc. Natl. Acad. Sci. USA 83:8231 (1986),and Orr et al. Proc. Natl. Acad. Sci. USA 83:1369 (1986) describenuclear transplantation experiments to activate Rana pipiens nuclei.DiBerardino was able to obtain tadpoles having a survival rate of up toa month, by transplanting differentiated somatic cells into enucleatedeggs.

[0077] The present invention discloses methods and products useful inactivating a non-dividing nucleus, and studying such activation. Thesemethods and products are especially useful for analyzing a nucleus fromnon-dividing human fetal cells such as aminocytes, keratinocytes,trophoblasts, erythrocytes and leukocytes. However, the methods andproducts are also useful for activating the nuclei from other types ofnon-dividing human cells such as other types of non-dividing fetal cellsand sperm, and non-dividing cells isolated from other mammals.

[0078] Preparing a nucleus for nuclear activation and bringing aboutnuclear activation is described in detail below as four differentphases: (1) preparation of non-dividing human nuclei, (2) preparation ofactivating egg extracts from a source such as activated Xenopus eggs,(3) preparation of non-activated CSF extracts from a source such asnon-activated Xenopus eggs, and (4) activation of non-dividing humannuclei.

[0079] Also described in detail below are modified CSF extracts whichcan bring about nuclear swelling in the absence of an activating eggextract; new procedures of pretreating a sperm cell to enhance itsactivation; a microchamber microscope slide which facilitates bringingabout nuclei activation and analysis of nucleic acids in such cells; akit for bringing about nuclear activation; an activation assay; and aprocedure for cloning whole organisms from somatic cell nuclei.

[0080] The featured methods and products can be used to cause activationof a non-dividing human nucleus thereby inducing swelling, and/or DNAreplication and/or the formation of metaphase chromosomes. Theprocedures provided herein regarding nuclei activation are generallybased upon existing procedures used in other systems. However, severalimprovements over the existing systems are disclosed. Furthermore,existing procedures have not previously been used on human fetal cellsnor was it known if they would produce useful results on such cells.

[0081] Examples are given to illustrate different aspects andembodiments of the present invention. It is to be understood thatvarious different modifications are possible and are contemplated withinthe true spirit and scope of the appended claims. There is no intention,therefore, of limitations to the exact process or disclosure hereinpresented.

[0082] In particular, there is shown below the activation of a humanfetal red blood cell nucleus using frozen/thawed activating egg extractwithout further pretreatment, under non-dividing conditions. The treatednuclei swelled significantly, replicated DNA, and then entered andarrested in the pre-mitotic state. Such nuclei are useful for prenataldiagnosis. Furthermore, the use of further pretreatment, should increasethe rate and extent of nuclear swelling, decrease the time it takes forDNA synthesis to occur after activation, increase the rate and extent ofDNA synthesis, and improve the efficiency with which metaphasechromosomes are formed.

I. NUCLEAR ACTIVATION

[0083] (1) Preparation of Nuclei

[0084] The present invention provides a method for activationnon-dividing mammalian cell nuclei, preferably non-dividing human cellnuclei. Before being activated non-dividing human nuclei are isolatedand pretreated. A preferred source of non-dividing human cells are fetalcells recovered from the blood of pregnant women such as trophoblasts,erythrocytes and leukocytes (such as granulocytes, neutrophils,basophiles and eosinophils). Isolating these cells does not requirepenetration of the womb. The present invention is also useful foranalyzing other types of non-dividing human cell nuclei, includingnon-dividing keratinocytes (e.g., those isolated from amniotic fluid),aminocytes, and sperm cells, or similar cells obtained from mammalsother than humans.

[0085] Non-dividing fetal cells can be recovered from maternal bloodsupply using techniques such as antibody staining followed by cellsorting. (For example, see Bianchi entitled Non-Invasive Method ForIsolation and Detection of Fetal DNA” PCT/US90/06623, herebyincorporated by reference herein). Antibody cell sorting techniquesseparate fetal and maternal cells based on the presence of differentantigens on fetal and maternal cells. The antigen can be differentiatedby suitable antibodies. Such antibodies which can be obtained by oneskilled in art include HLe-1 which recognizes an antigen present onmature human leukocytes, such as granulocytes, and very immatureerythrocyte precursor but not nucleated cells, and antibodies to thetransferrin receptor. (E.g., see Bianchi, supra PCT/US90/06623.)Procedures using antibodies can be carried out by contacting a samplecontaining fetal and maternal blood with a labeled antibody recognizingeither fetal cells or maternal cells. The antibody labeled cell can besorted using standard techniques including flow cytometry,immunomagnetic beads and cell panning.

[0086] Non-dividing human cells should be isolated under mild conditionsdesigned to prevent activation of extracellular proteases (for instancethose of the plasma), intracellular proteases, or nucleases, as well asto prevent mechanical damage to cell structures. Inadvertent protease ornuclease activation during nuclear isolation could result in damagingboth the genetic material of the cell and the protein structures withinor around the nucleic acid. Possible nucleic acid damage includes,nucleic acid degradation, and damage to the structural state (e.g.,supercoiling). One advantage of keeping the protein structure intact, ismaintaining the cytoskeletal protein so it can be subsequently separatedto from nucleic acid under mild conditions minimizing damage to histonesand non-skeletal proteins.

[0087] Preferably, solutions used to isolate cells contain proteaseinhibitor. Solutions used to isolate cells such as HBSS and NIBsolutions can be supplemented with protease inhibitors as follows: 0.1mg/ml heparin, 0.1 MM TPCK (N-tosyl-L-phenylalanine chloromethylketone), 0.1 mM TLCK (Nα-p-tosyl-L-lysine chloromethyl ketone), 0.05 mMPMSF (phenylmethylsulfonyl fluoride), 5 μg/ml leupeptin, or 31.25 mMNa₂S₂O₅.

[0088] After cell purification, the cell nucleus is preferably isolatedand pretreated under mild conditions. Nuclear pretreatment is preferablycomprised of two steps, which may be carried out simultaneously orseparately; (1) membrane permeabilization, and (2) separation oralteration (e.g., denaturation and degradation) of cytoskeletal proteinsand nuclear matrix proteins. Treatment should be carried out to minimizethe damage to nucleic acid within the nucleus.

[0089] The separation or alteration of certain protein appears to be anecessary step for activation. In Xenopus erythrocytes, for instance,proteolytic digestion of cytoskeletal proteins, such as vimentin,appears to be a necessary step for subsequent nuclear activation.Coppock et al. Developmental Biology 131:102 (1989). The pretreatmentshould prepare the nucleus for subsequent activation rather than causeactivation.

[0090] Desired conditions for plasma membrane permeabilization includemild detergent treatment, mild protease treatment, mild shearing, andmild hypotonic shock. Mild conditions are those conditions able topermeabilize the plasma membrane while creating the least amount ofdamage to the nuclear DNA and proteins. Permeabilization can be detectedusing trypan blue. Trypan blue is a dye which cannot enter intact cells.The entrance of trypan blue into a cell indicates permeabilization.Protein degradation due to inadvertent protease activation can bedetermined using polyacrylamide gel electrophoresis to look for proteindegradation products. The intactness of nuclear nucleic acids can beestablished by using agarose gel electrophoresis to determine thepresence of nucleic acid degradation products.

[0091] Possible pretreatments for separation or alteration ofcytoskeletal proteins and nuclear matrix proteins include the following:

[0092] (a) Treatment with one or more thiol reducing agent such as 10 mMdithiothreitol for a limited time, at a controlled temperature and pH,to denature cytoskeletal protein;

[0093] (b) Controlled salt extraction, such as by washing in bufferssupplemented with increasing amounts of NaCl or KCl in the range of0.025 to 1.0 M, to selectively remove cytoskeletal proteins and proteinsbound to DNA;

[0094] (c) Controlled poly-anion treatment, such as heparin at 0.01-1.0mg/ml or penta sodium tripolyphosphate at 70 mM, in 10 mM borate buffer(TPP) at pH 9.0, to selectively remove positively charged cytoskeletalprotein;

[0095] (d) Degradation of cytoskeletal proteins using a protease.

[0096] The extent of protein and DNA damage can be measured as describedabove. Preferably, nuclear isolation and pretreatment are bothaccomplished at the same time using mild concentrations of lysolecithin(e.g., 40 μg/ml) and protease (e.g., 0.3 μg/ml trypsin), such that aminimal amount of damage to non-cytoskeletal proteins, histones, andnucleic acid occurs. The minimum time and temperature required fordetergent and protease treatment should be used. In the case of redblood cells this is about 10 minutes at 25° C., using 0.3 μg/ml oftrypsin and 40 μg/ml of lysolecithin. As would be appreciated by oneskill, the preferred time and temperature will change as theconcentration of the reagents change.

[0097] Controlled treatment with ion-selective chelating agents may alsobe performed as an additional pretreatment. Suitable ion-selectivechelating agents include EGTA which can chelate Ca²⁺, EDTA which canchelate Ca²⁺ and Mg²⁺, and mimosine which can chelate of Cu²⁺, Al³⁺, andFe³⁺. These ions stabilize higher order chromatin structure, thus theirchelation may aid in chromatin decondensation.

[0098] Methods to terminate the detergent and protease pretreatmentinclude adding proteins to adsorb detergents (such as 0.4% bovine serumalbumin, the bovine serum albumin employed at this step should beprepared by dialysis of commercially available BSA fraction V againstdistilled water to remove soluble salts followed by lyophilization), andadding protease inhibitors (such as soybean trypsin inhibitor) to thereaction. The pretreated nuclei should be subsequently washed using anice cold solution designed to preserve genomic DNA intactness. NIBbuffer can be used for this purpose. NIB is made up of 250 mM sucrose,25 mM NaCl, 10 mM Pipes, 1.5 mM MgCl₂, 0.5 mM spermidine, and 0.15 mMspermine, pH 7.0.

[0099] The overall efficacy of mild conditions to obtain a pretreatednucleus can be determined by: a) microscopic examination of nuclei toassess whether nuclei are free of their surrounding cytoskeleton and arefree standing or clumped, clumping of nuclei is a strong indication ofnuclear damage since many nuclei get trapped in released DNA; and b) theability of nuclei to respond to activating egg extract, the use of mildconditions increases subsequent activation of individual nuclei andimproves the synchrony and homogeneity with which the entire populationof nuclei is activated.

[0100] (2) Preparation of Activating Egg Extract

[0101] Activating egg extracts can be used to cause non-dividing nucleito swell, assemble nuclear envelopes and lamina, replicate theirgenomes, enter mitosis, and form metaphase chromosomes. Activating eggextracts contain material, such as precursors, protein(s), nuclearmembrane vesicles, or mRNA required to activate non-dividing nuclei.

[0102] Non-activated eggs can be triggered en masse to produce materialwhich brings about activation, by being chemically induced to enter thecell cycle. Eggs can be induced using standard techniques such aselectric shock, pricking with a needle, fertilization and the use of acalcium ionophore. (See, Gerhart, et al., J. of Cell Biology 98:1247,1984, and the procedures described below.) The induced eggs enter intothe cell cycle. It appears that when an egg is at the point in the cellcycle just prior to the S-phase, the egg cytoplasm is most active insupporting activation. As the egg proceeds into and past the S-phase, itappears to produce material inhibitory to nuclear activation (see Table1).

[0103] One of the benefits of the disclosed procedures is obtaining anactivating egg extract having a higher DNA synthesis activation activitythan activating egg extract disclosed in the prior art. The DNAsynthesis activation activity can be determined by measuring thesynthesis of DNA using labelled precursors.

[0104] Hardened Xenopus eggs are a good source for preparing anactivating egg extract. Hardened Xenopus eggs are stable for severalhours. In contrast, “soft” Xenopus eggs must be used rapidly. As soon assoft eggs are dejellied they tend to induce spontaneously and randomly.This is considered disadvantageous because activating egg extractsprepared from a specific time during the cell cycle, just prior to theS-phase, have a higher DNA synthesis activating activity than extractsprepared from other phases of the cell cycle. Thus, it is desirable tosynchronously induce a large number of eggs which are all at the samepoint of the cell cycle, so extracts can be prepared later from a largenumber of eggs all of which have elevated DNA synthesis activationactivity at the same time.

[0105] Freshly ovulated Xenopus eggs can be hardened by stabilizing theeggs vitelline envelope as described by Wangh, J. Cell Science 93:1(1989). Obtaining freshly ovulated eggs from female Xenopus isfacilitated by injecting hormones which cause Xenopus to ovulate.Injecting 600 units of human chorionic gonadotropin (HCG) into a Xenopusfemale generally brings about ovulation within 12-15 hours. Injection ofpregnant mare serum gonadotropin about 24 hours before HCG treatmentsignificantly increases the yield of mature eggs. Furthermore, repeatedovulation of frogs once every 4-8 months improves the yield of eggs byincreasing the synchrony of oocyte development in the ovary.

[0106] The freshly ovulated eggs within their jelly coat, are floodedwith 0.3×NKH (1×NKH is 40 mM NaCl, 2.5 mM KCl, 7.5 mM Hepes, pH 7.4 withNaOH), for 15-20 minutes. During this time the jelly layers swell. Theeggs are then dejellied in 3×NKH containing 2% cysteine, pH 7.9, bygentle swirling for about 5 minutes.

[0107] The resulting soft eggs can be “hardened” by immediately rinsingthem five times in 3×NKH containing 2 mM MgCl₂, 1 mM CaNO₃, 10 μM ZnCl₂and letting them stand for at least 20 minutes at room temperature.Hardened eggs are sorted to remove damaged and partially induced eggs.Calcium is required for hardening and must be present in the 3×NKH usedto wash cysteine-treated eggs. Eggs washed in the absence of Ca²⁺ andsubsequently treated with Ca²⁺, and Ca²⁺ treatment of eggs still in thejelly. coat, do not result in hardened eggs. Additionally, adding Ca²⁺before or during dejellying will not result in hardening.

[0108] Activating egg extract is preferably obtained from hardened eggsinduced en masse. Induction can be carried according to proceduresdescribed by Coppock et al., Developmental Biology 131:102 (1989). Theprocedure described by Coppock et al. as modified, in the followingmanner, was used to obtain “prepared activating egg extract”: 5-15 ml ofhardened eggs were rinsed using activation buffer (4 mM NaCl, 0.14 mMpotassium gluconate, 2 mM Hepes, 2 mM MgSO₄, and 0.6 mM Ca(NO₃)₂, pH7.8), and placed in 500 ml of activation buffer; the eggs were theninduced by adding calcium ionophore A23187 (10 μM in DMSO; SigmaChemical Co.) to a final concentration of 100 nM and incubating at roomtemperature; after 10 minutes calcium ionophore treated eggs were rinsedand induced for an additional 15-20 minutes by incubating in 1.5×NKHcontaining 2 mM MgCl₂, and 0.6 mM CaCl₂ (Coppock et al., supra, stopsthe induction at 10 minutes); the eggs were then placed on ice in asiliconized or teflon beaker and washed 3-5× in several hundredmilliliters of ice cold EB buffer (EB=50 mM potassium gluconate, 250 mMsucrose, 10 mM potassium HEPES, 1.5 mM MgCl₂, pH adjusted to 7.5 withpotassium hydroxide); eggs were then transferred to a volumetricpolyallomer centrifuge tube, mixed with Versilube F-50 oil (GeneralElectric) at 0.2 ml oil/ml eggs, and tight packed by centrifugation at40×g for 1 minute, at 2-4° C.; the overlaying oil and aqueous layerswere removed and the eggs were crushed by centrifugation 15 minutes at9000×g, at 2-4° C; the cytoplasmic layer between the yolk pellet and theoverlaying lipid layer was collected from the bottom by puncturing thetube with a syringe needle; cytochalasin B was added to a finalconcentration of 10-50 μg/ml and the cytoplasmic material recentrifugedfor 15 minutes at 9,000×g, at 2-4° C.; the resulting second cytoplasmicsupernatant was recovered and either used fresh or frozen for futureuse.

[0109] This procedure for “prepared activating egg extract” involving anincreased induction time of 15-20 minutes over that described in Coppocket al. supra, was chosen based upon the following two experiments: 1)plasmid DNA injected into non-activated Xenopus eggs does not beginreplicating until 25-30 minutes after eggs are induced, during this lagperiod factors required for DNA synthesis are possibly released,altered, or synthesized within the egg; and 2) extracts prepared fromeggs induced for only 10 minutes synthesize additional proteins in vitrowhich first act to increase DNA synthesis in pre-treated Xenopuserythrocyte nuclei and then act to inhibit DNA synthesis in these samenuclei.

[0110] The second experiment, “induction optimization,” is particularlyuseful in determining the optimal induction time for obtainingactivating egg extract having an elevated DNA synthesis activationactivity. The experimental results for induction optimization used toobtain an activating egg extract with an elevated DNA synthesisactivation activity from Xenopus, is shown in Table 1. The sameexperimental design could be used to establish the induction time neededto obtain egg extracts having elevated DNA synthesis activation activityfrom species other than Xenopus.

[0111] Induction optimization was carried for Xenopus as describe below(see also Example 2, infra, for further optimization experiments).Xenopus erythrocyte nuclei were isolated and pretreated withlysolecithin and trypsin as described in Example 2 (described below). Anactivating egg extract was prepared from hardened eggs which wereinduced for 10 minutes as described above. Both the activating eggextract and the pretreated nuclei were kept on ice (about 4° C.). Theactivating egg extract was supplemented with 1 mM ATP (not used in theother examples described herein), 10 μg/ml creatine phosphokinase, 10 mMcreatine phosphate, 10 μCi P³²-dCTP and combined with pretreated nuclei(about 200 nuclei/μl). Individual aliquots containing activated nucleiwere shifted from 4° C. to 25° C. Cycloheximide to a concentration of100 μg/ml was added to the individual aliquots at different times. Thealiquots were then incubated at 25° C. for a total time, including thetime at 25° C. before addition of cycloheximide, of 60 minutes. After 60to minutes, P³²-dCTP incorporation into newly synthesized DNA wasdetermined. TABLE 1 Time CHM Added (Minutes) Cpm Incorporated into DNANo CHM 1,296  0 2,894  5 4,208 15 4,937 30 3,775 45 2,314

[0112] The result of induction optimization for Xenopus activating eggextract is shown in Table 1. The highest observed DNA synthesisactivation activity was 15 minutes after the addition of cycloheximide.Thus, about fifteen minutes appears to be the additional time requiredfor peak DNA synthesis activation activity (total induction time ofabout 25 minutes). A more precise time point for the activation peak maybe readily determined by taking additional experimental time points.Elevated DNA synthesis activation activity (more DNA synthesisactivation activity than zero time), was seen after an additional 5, 15,and 30 minutes. The elevated DNA synthesis activation activity decreasedfrom 15 to 30 minute time points. After the 45 minute time point theobserved DNA synthesis activation activity was below that of theelevated DNA synthesis activation activity. The decrease in DNAsynthesis activation activity observed for incubation times longer thanan addition 15 minutes is attributed to the synthesis of proteinsinhibitory to activation.

[0113] As would be appreciated by one skilled in the art, the optimalDNA synthesis activation time will also vary as the temperature changes.As the temperature increases the optimal DNA synthesis activation timedecreases, however, the temperature is preferably not raised above 24°C. As the temperature decreases the optimal DNA synthesis activationtime increases, however, the temperature is preferably not lowered below16° C.

[0114] Several proteins present in Xenopus egg extracts are involved inDNA replication. One or more of these could be a positive acting proteinsynthesized during the first 25-30 minutes after activation responsiblefor the increase in DNA synthesis activation activity. Possible positiveacting proteins include: cyclin A, RFA single strand binding protein,cdk2 kinase, and RCC1 protein. There are also several proteins whosesynthesis after the first 25 minutes could be responsible for thedecrease in DNA synthesis activation activity. Possible proteins whichcould decrease DNA synthesis activation activity include cdc2 and cyclinB. Given the evolutionary conserved nature of both the positive andnegative acting proteins, and their functions, it is likely that eggsfrom species other than Xenopus also display an optimal time just beforethe start of S-phase when their cytoplasm is most active in supportingDNA synthesis activation.

[0115] As seen in Table 1, incubating for 10 minutes provided less than60% of the peak DNA synthesis activity observed compared to the optimalDNA synthesis activity of extracts prepared from Xenopus eggs. Using thetechniques described herein, the induction time required for obtainingegg extract having an elevated DNA synthesis activation activity (morethan 70% of the peak activation) can be obtained for activating eggextract prepared from egg sources other than Xenopus.

[0116] Activating egg extracts from “hardened” eggs may be used fresh inwhich case they support more than one cell cycle in vitro. Alternately,these extracts may be frozen and then thawed, in which case they areable to support one or more cell cycles in vitro.

[0117] The activating egg extract is preferably made 7.5-10% (v/v) inglycerol and stored frozen in liquid nitrogen, by standard techniques orby an increased rapid freezing technique. The increased rapid freezingtechnique freezes the extract faster than merely suspending in liquidnitrogen. Increased rapid freezing can be achieved by spotting extract,made 7.5-10% (v/v) glycerol, as 20 μl droplets onto a block of aluminumimmersed in liquid nitrogen.

[0118] Before use, frozen activating egg extracts are thawed rapidly atroom temperature, put on ice, and if desired, supplemented to enhanceactivation activity. One possible supplement is cyclic-AMP. The additionof 0.1 mM to 10 MM cAMP to activating egg extracts increases theactivation activity of the activating egg extract, as measured bysubsequent DNA replication in pretreated Xenopus erythrocyte nuclei.cAMP can be broken down by phosphodiesterase. Caffeine is an inhibitorof phosphodiesterase and, thus, enhances the stability of endogenous andadded cAMP. Thus, caffeine and phosphodiesterase inhibitors are possiblesupplements to enhance activation activity of activating egg extract.Indeed, the addition of caffeine to activating egg extract was found toincrease subsequent DNA replication in activated Xenopus nuclei.

[0119] Appropriate egg extracts can be obtained from sources other thanXenopus. Useful guidelines for choosing an appropriate egg source tomake either activating egg extract or CSF extract are provided below.These guidelines are not intended to be a list of requiredcharacteristics, but rather a list of considerations useful for choosingan egg source.

[0120] Useful guidelines for choosing an appropriate egg source formaking egg extracts include the following:

[0121] 1. Egg/embryo with substantial stores of activating cell cyclematerial are preferred. Such egg/embryos can be identified as thoseshowing a series of rapid cell cycles, i.e., cell divisionsapproximately once every hour as compared to once every day.

[0122] 2. Moderate egg size is preferred. Moderate egg size represents acompromise between the cytoplasmic volume per egg and yolk mass per egg.Preferably a large yield of cytoplasm per volumetric measure of eggs isobtained.

[0123] 3. A species in which female animals shed a large number of eggsis preferred as a means of increasing the amount of egg extractavailable from an animal, while keeping the cost of caring for theanimal at a minimum. However in some instance, such as the activation ofmammalian somatic cell nuclei prior to transplantation into theircorresponding eggs, it may be desirable to prepare extracts frommammalian eggs despite their small size and relatively small number perfemale.

[0124] 4. Females of the chosen species are preferably identifiable byexternal characteristics.

[0125] 5. Females preferably breed in a reasonable period of time (atleast once per year), and at a reasonable cost.

[0126] 6. Eggs are preferably shed as single cells (e.g., not in jellymass), or easily freed of jelly layers and other major externalenvelopes.

[0127] 7. Eggs can preferably be stabilized from activating once freedof extracellular coats. (see e.g., Wangh, J. Cell Science 93:1 (1989)).

[0128] 8. Females preferably produce high quality eggs which are uniformand regular. These features minimize waste and help in developingautomated methods to sort good and bad eggs. Some eggs, such as those ofechinoderms and mollusks, are transparent and contain a prominentgerminal vesicle nucleus which can be used to judge egg quality. Othereggs, such as those of Xenopus, are not transparent, but have twodistinct colors which can also be used to judge egg quality.

[0129] 9. Eggs are preferably chemically inducible in a synchronousmanner such that a number of eggs may be induced at the same time and beapproximately at the same point in the cycle at a specified later time(preferably at meiotic metaphase state or mitotic metaphase state). Inthis way, extracts may be obtained from a number of eggs at the samepoint in the cell cycle by inducing all the eggs at one time and usingall the eggs to prepare an extract at a later time.

[0130] 10. Females can preferably be chemically induced to ovulatethereby making it possible to increase the production of eggs from agiven female.

[0131] 11. Female are preferably not harmed by the egg collectionmethod. Alternately, if egg collection does harm the female thosefemales for which a commercial use of the carcass exists are preferred.

[0132] 12. Preferably the eggs allow preparation of extracts that inducenuclear swelling, either without or with concomitant DNA replication.Nuclear swelling without replication can be achieved by removal ofmembrane vesicles required for nuclear envelope assembly, or byinhibition of DNA synthesis (e.g., using reagents such as aphidicolin,mimosine, or DMAP), or by CSF extract supplemented with a kinaseinhibitor (e.g., such as DMAP or staurosporine). Nuclear swelling withreplication can be achieved using activating egg extracts such as thoseobtained from Xenopus eggs.

[0133] 13. It is important that eggs used to prepare CSF extracts can bearrested in either the meiotic metaphase state, or in the mitoticmetaphase state. Recovery of chromosomes, rather than interphase nuclei,requires cell cycle arrest in metaphase. For some species, extracts inmetaphase arrest can be prepared directly from non-activated eggs, suchas unfertilized Xenopus eggs, or can be made to cycle into and arrest inmeiotic metaphase. Useful reagents for bringing about and causing arrestin meiotic metaphase include cyclin

90 (a non-degradable form of sea urchin cyclin), other cyclin relatedpeptides, small amounts of CSF extract (prepared from non-activatedXenopus eggs), components found in non-activated Xenopus eggs (such asc-MOS kinase) or Calyculin A used on echinoderm eggs (Tosuji et. al.Proc. Natl. Acad. Sci. 89:10613 (1992)).

[0134] (3) CSF Extract Treatment of Nuclei

[0135] Non-activated CSF extract can be used to aid subsequent nuclearactivation of non-dividing mammalian cell nuclei, including human cellnuclei, without directly causing nuclear swelling or DNA replication; orto directly cause nuclear swelling as discussed in section II infra.Nuclei in CSF extract appear to condense into chromosome like structuresand may become surrounded by a spindle apparatus. Nuclear activationprior to contact with an activating egg extract is disadvantageous.Problems with premature activation include a decrease in the enhancementof activation and different nuclei being activated at different times.

[0136] The ability of CSF extract to enhance activation may be increasedby various supplement. In addition, the incubation conditions of nucleiin CSF extract can be adjusted to improve the ability of such extractsto enhance activation of the nuclei upon subsequent contact withactivating egg extract.

[0137] The CSF extract is preferably prepared from non-induced eggsarrested at meiotic metaphase. CSF extract prepared from non-inducedeggs arrested at meiotic metaphase contain high levels of mitosispromoting factor (MPF) activity and cytostatic factor (CSF) activity.CSF and MPF are factors present in CSF extract which are believed to aidin subsequent activation of quiescent nuclei by altering cytosketalproteins, nuclear matrix proteins, and nuclear histones, particularly byphosphorylation of these proteins.

[0138] MPF is an activity controlling nuclear entry into mitosis andinitiation of spindle assembly. MPF is composed of two catalyticsubunits, p34^(cdc2) and cyclin B. At the onset of anaphase, cyclin B isdestroyed resulting in the inactivation of MPF. During anaphase thechromosomes move towards the two opposite poles of the spindle apparatusand subsequently decondense.

[0139] CSF is an activity responsible for metaphase arrest inunfertilized vertebrate eggs. CSF activity is due to at least twokinases: mitogen-activated kinase (MAP) and cdk2/cyclin (cdk2 is akinase related to cdc2, but the regulatory subunit of cdk2 is cyclin E(or A) rather than cyclin B). The activities of MAP appears to becontrolled by additional kinases such as c-Mos kinase.

[0140] One reason for obtaining CSF extract from eggs arrested atmeiotic metaphase, is that both MPF and CSF are inactivated uponinitiation of the cell cycle.

[0141] CSF extracts from non-induced Xenopus eggs can be prepared by amethod based on the work of Lohka and Masui, Developmental Biology103:434 (1984), as well as that of Murray et al., Nature 339:280 (1989).A procedure for obtaining CSF extract is as follows. Eggs are obtainedfrom one or more ovulating frogs as described above. Each batch offreshly ovulated eggs, about 500 to 1000 eggs, is hardened as describedabove. Damaged and activated eggs are removed. The remaining eggs arecombined into a large siliconized glass or teflon beaker and washed 4-5times at room temperature (about 21° C.) in approximately 500 mlEB-buffer containing 5 mM potassium EGTA, pH 7.5, (EB=50 mM potassiumgluconate, 250 mM sucrose, 10 mM potassium HEPES, 1.5 mM MgCl₂, pHadjusted to 7.5 with potassium hydroxide). The eggs are then transferredto a volumetric polyallomer centrifuge tube, mixed with Versilube F-50oil (General Electric) at 0.2 ml oil/ml eggs, and are tight packed bycentrifugation at 40×g for 1 minute, at room temperature. The overlayingoil and aqueous layers are removed and the eggs are crushed bycentrifugation for 15 minutes at 9,000×g, at 2-4° C. The cytoplasmiclayer between the yolk pellet and the overlaying lipid layer iscollected from the bottom by puncturing the centrifuge tube with asyringe needle. Cytochalasin B is added to a final concentration of10-50 μg/ml and potassium EGTA is added to a final concentration of 1mM. The cytoplasmic material is mixed by gently pipetting or rockingback and forth, the cytoplasmic material is then centrifuged for 15minutes at 9,000×g, at 2-4° C. An alternative centrifugation procedureinvolves preparation of a high speed supernatant by centrifugationat>100,000×g for 2 hrs at 2-4° C. In either case, the resulting secondcytoplasmic supernatant (hereinafter “prepared CSF extract”) isrecovered and is either used fresh or is made 7.5-10% in glycerol andfrozen for future use in the same manner as activating egg extract.Preferably, CSF extract is incubated at 25° C. for 2 hours prior tofreezing. The level of histone H1 kinase activity increases several foldduring the period of incubation.

[0142] Frozen extracts can be used by thawing rapidly at roomtemperature and then placing on ice. Thawed extracts are preferablysupplemented with an ATP regenerating system consisting of 10 mMcreatine phosphate, and 10 μg/ml creatine phosphokinase.

[0143] The histone H1 kinase activity, the structural state of plasmidDNA added to the CSF extract, and the inability of CSF extract to causenuclear activation, demonstrated that “prepared CSF extract” wasarrested in meiotic metaphase. The histone H1 kinase activity of the CSFextract either before or after freezing was high. Upon activation of theextract with 1.2 to 4 mM Ca²⁺, the histone activity decreased.Preferably, 1.2 mM Ca²⁺ is used when CSF extract is supplemented with 1mM EGTA to achieve recycling. No recycling occurs when 3 to 4 mM of Ca²⁺is used in CSF extract supplemented in the presence of 1 MM EGTA.Negatively supercoiled circular plasmid DNA added to the extractrelaxed. Lysolecithin-trypsin pretreated Xenopus erythrocyte nucleiadded to CSF extract failed to swell or synthesize DNA.

[0144] After further pretreatment in CSF extract, the nuclei may beactivated by adding 9 volumes of “prepared activating egg extract.” DNAreplication, measured by incorporation of labelled nucleotides into DNAstrands, may be used to determine the extent to which prior treatment inCSF extract enhances nuclear activation in activating extract. Labellednucleotides useful in measuring nuclear DNA replication includemicrocurie amounts of P³²-dCTP for radioactive measurement of newlysynthesized DNA, 16-50 μM biotinylated-dUTP or BrdUTP for fluorescentmeasurement of newly synthesized DNA, and 250 μM BrdUTP for densitylabelling of newly synthesized DNA.

[0145] Several supplements to CSF extract, in the proper concentration,increased the ability of CSF extract to enhance activation activitywithout resulting in premature DNA synthesis activity. Usefulsupplements include μ-glycerol-PO₄, Ca²⁺, and protein kinase inhibitors.The addition of β-glycerol-PO₄ increased the rate at which negativelysupercoiled DNA relaxed in CSF extract and was subsequently assembledinto chromatin. A concentration of about 80 mM β-glycerol-PO₄ was foundto aid in chromatin assembly without causing DNA synthesis.Beta-glycerol-PO₄ is an inhibitor of phosphatase activity and may act byincreasing the level of the phosphorylated functionally-active form oftopoisomerase II in the CSF extract.

[0146] Similarly, the addition of 100 μM Ca²⁺ increased both the ratenegatively supercoiled DNA relaxed in CSF extract and rate of subsequentassembly into chromatin. Calcium is a cofactor for calcium calmodulinactivated protein kinases and may also act by increasing the level ofphosphorylated active topoisomerase II activity in the CSF extract. Theaddition of 100 μM CaCl₂ to thawed CSF extract failed to trigger itsentry into the cell cycle as judged by continued high levels of histoneH1 kinase activity. The addition of 100 μM Ca²⁺ also increased both theamount and the rate of DNA synthesis in erythrocyte nuclei afteraddition of activating egg extract. CSF extract responded to theaddition of 1.2-4 mM Ca²⁺ by increasing the rate and extent of chromatinassembly over that seen upon addition of 100 μM Ca²+. However, thehigher concentration of calcium also activated the CSF extract.

[0147] The association of factors, whose presence increase DNA synthesisactivity of CSF extract was also examined. Apparently, one or morefactors in CSF extract which aid in subsequent DNA replication areloosely held by the nuclei in CSF extract and are lost during washing.Xenopus cell nuclei were pretreated with trypsin and lysolecithin, addedto CSF extracts to a concentration of 1000-2000 nuclei per μl, andeither washed by diluting into excess NIB buffer and centrifuging, ornot washed. Subsequent addition of activating egg extract, to aconcentration of 100-200 nuclei per μl, resulted in less DNA replicationfor washed nuclei. For this reason, CSF extracted treated human nucleiare preferably not washed prior to contact with activating egg extract.

[0148] Nuclear activation upon contact with activating egg extract canbe increased by manipulating the conditions in which nuclei areincubated in CSF extract during further pretreatment. Usefulmanipulations can be obtained by regulating the incubation period andtemperature. The use of a warm-then-cold regime stimulates subsequentnuclei activation. Both warm and cold steps appear to exert positiveeffects on subsequent nuclei activation. Preferably, the warm-then-coldregime comprises incubation at about 25° C. for 30-90 minutes followedby incubation at 4° C. for 30-90 minutes.

[0149] Trypsin and lysolecithin treated Xenopus red blood cell nucleiincubated in frozen/thawed CSF extract using a warm-then-cold regime andcontacted with fresh activating egg extract resulted in extensive andsynchronous nuclear envelope formation, swelling, and replication uponcontact with freshly prepared activating egg extract. While this systemis attractive to aid in activation of human nuclei, because of theconvenience of using frozen CSF extract, use of this system on Xenopuserythrocyte nuclei revealed several limitations. One limitation is theneed to freshly prepare activating egg extract, which is experimentallyinconvenient.

[0150] Using CaCl₂ in conjunction with frozen CSF overcomes thislimitation. The use of CaCl₂ permits synchronous nuclear envelopeformation, swelling, replication, entry into mitosis (includingformation of chromosome-like structures without DNA fragmentation), andrenewed DNA synthesis in a second S-phase when both frozen CSF andfrozen activating egg extracts are used. Thus, the use of both awarm-then-cold regime and CaCl₂ is particularly advantageous when frozenactivating egg extracts and frozen CSF extracts are used to causenucleus activation. Preferably the CSF extract contains 0.1 to 0.4 mMCaCl₂ to enhance nuclear activation upon subsequent contact withactivating egg extract. At this range of Ca²⁺, nuclei treated in CSFextract should not activate until contact with activating extract.

[0151] (4) Activation of Nuclei With Activating Egg Extract

[0152] Activating egg extracts can be used to activate non-dividingmammalian cell nuclei, such as non-dividing human cell nuclei, to bringabout swelling, chromatin decondensation, DNA replication and formationof metaphase chromosomes. However, nuclear activation can be stopped atvarious points and information about nucleic acid sequence and structurecan be obtained by examining the resulting DNA. Under duplicationconditions,the nucleus swells, DNA replicates, and the resultantchromosomes divide. Under non-duplication conditions, the nucleusswells, DNA is replicated, but the resultant chromosomes do not divide.Under non-synthesis conditions the nucleus swells, but DNA is notreplicated, and nuclei do not divide.

[0153] The use of nocodazole, or other drugs like colchine, colcemid,and D₂0 which inhibit microtubule assembly is preferred for preventingseparation of mitotic chromosomes. These drugs prevent the formation ofmitotic spindles during the cell cycle. As a result, condensedchromosomes accumulate rather then separate to the cells poles and arereadily visualized for karyotypic analysis.

[0154] However, the addition of 5 μg/ml nocodazole to activating eggextract decreases the rate of DNA replication. Thus, to maintain a highrate of DNA replication it is necessary to either: 1) use nocodazole ata dose less than 5 μg/ml, such as adding nocodazole to CSF extract at 5μg/ml and diluting the mixture with 9 volumes of activating egg extract;2) use another drug such as colchine, colcemid or D₂O (deuterium oxide)which may be able to block mitotic spindle formation without inhibitingDNA replication; or 3) add the spindle inhibitor later, i.e., after DNAsynthesis is complete but before nuclei proceed into mitosis.

[0155] To avoid artifacts such as chromosome fragmentation duringnuclear activation it is desirable that complete, rather than partial,replication of nuclear genomes be achieved. The following techniques areuseful to assess the extent of genome replication achieved duringnuclear activation:

[0156] 1) Coordinate observations of the kinetics of DNA synthesis, thesize of the DNA molecules made, the timing of mitosis following DNAsynthesis, and the morphological appearance of nuclei. Completereplication is characterized by a early onset and rapid rate of DNAsynthesis in all nuclei, an abrupt cessation of DNA synthesis in allnuclei, followed by rapid entry into mitosis, and renewed replicationwhen nuclei exit mitosis. In addition, newly synthesized DNA moleculesare very long (greater than 50,000 base pairs), but are transientlycleaved by type II topoisomerase during the period of chromosomecondensation and decondensation.

[0157] 2) The isotope dilution technique can be used to measure the poolsize of DNA precursors in the activating egg extract to establish theextent of genome replication, on the basis of the radioactive specificactivity of the DNA. The isotope dilution technique can be carried outaccording to Blow and Laskey, Cell 47:577 (1986).

[0158] 3) BrdUTP density labelling of newly replicating DNA followed byisopycnic centrifugation in CsCl and Southern hybridization can be usedto determine if one or more rounds of replication is occurring. Duringthe initial round of semi-conservative replication, incorporation ofBrUTP leads to formation of a DNA duplex having one heavy (BrUTPcontaining) strand and one light strand. The subsequent production of aDNA duplex containing two heavy strands indicates more than one round ofreplication.

[0159] 4) DNA replication can be visually measured usingbiotinylated-deoxynucleotide triphosphates (such as biotin-11-dUTP) orbromodeoxy-UTP. These labeled nucleotides can be added to activating eggextracts and re incorporated into DNA during replication. Nucleicontaining the labelled DNA can be recovered and examined in afluorescent microscope. The labelled DNA is conveniently visualized bystaining with Texas Red streptavidin (for biotin samples) or FITC(fluorescein) anti-BrdUTP antibodies. Total DNA can be visualized usinga fluorescent intercalating dye (such as propidium iodide or Hoechststain) or a fluorescently tagged reagent. In some cases it may bedesirable to treat nuclei in the microchamber microscope slide with highsalt solutions to stretch the DNA across the glass surface before DNAstaining. A fluorescent microscope can be employed to establish whetherall regions of the nuclear DNA (stained for instance with Hoechst)contain newly synthesized DNA (stained for instance with biotin-TexasRed streptavidin)

II. USE OF A CSF EXTRACT TO CAUSE NUCLEAR SWELLING

[0160] Nuclear swelling can be brought about in non-dividing nuclei byusing a modified CSF extract, made by high or low speed centrifugation,or by using a CSF extract or modified CSF extract made by high speedcentrifugation (a “partially purified CSF extract”). Use of a CSFextract to induce nuclear swelling is preferably carried out on isolatednuclei pretreated to separate the nuclei from its surroundingcytoskeleton, preferably with detergent and a protease as describedherein. Preferably, the modified CSF extract is a partially purified CSFextract and is modified by either (a) diluting with an aqueous solutionand/or (b) supplementing with a protein kinase inhibitor. Fresh CSFextract or frozen thawed extract can be modified.

[0161] Dilution of CSF extract to enhance its ability to cause nuclearswelling may be carried out using various aqueous solutions such aswater and physiological pH buffers. The aqueous solution is preferablybuffered to about pH 6.5 to about pH 7.5. An example of an appropriatebuffer is EB buffer (EB=50 mM potassium gluconate, 250 mM sucrose, 10 mMpotassium HEPES, 1.5 mM MgCl₂, pH adjusted to 7.5 with potassiumhydroxide). Preferably, the aqueous solution is added in an amount toachieve 25% to 75% dilution.

[0162] The ability of CSF extract to cause nuclear swelling can also beenhanced by using a protein kinase inhibitor such as DMAP orstaurosporine. DMAP and staurosporine are broad range kinase inhibitorsable to inhibit the actions of both CSF and MPF. Other protein kinaseinhibitor able to inhibit CSF and/or MPF can be obtained by one skilledin the art. The chosen kinase inhibitor can preferably inhibit both CSFand MPF.

[0163] Preferably, 2.5-5 mM of DMAP is used. The use of protein kinaseinhibitors should block kinase activities in the extract, includinghistone H1 kinase and result in the treated nuclei forming envelopes butfailing to initiate DNA replication. The proper protein kinase inhibitorconcentration can be empirically determined by one skilled in the art bymeasuring the extent of swelling and DNA replication in the presence ofdifferent amount of protein kinase inhibitors.

[0164] Preferably, an aqueous solution and a protein kinase inhibitorare both used to modify CSF extract. Nuclei treated with diluted CSFextract supplemented with DMAP (CSF-DMAP) swell to a greater volume thannuclei treated with undiluted CSF-DMAP. Preferably, CSF containing aprotein kinase inhibitor (CSF-PKH) is diluted 25% to 75% using anappropriate buffer and nuclei are incubated for more than 60 minutes at25° C., and more preferably around 90 minutes at 25° C. prior tomeasuring nuclei swelling.

[0165] Diluted CSF-PKH extract can be further modified by altering theCa²⁺ and Mg²⁺ ion concentration to further increase swelling, and affectchromatin condensation and decondensation. Ca²⁺ and Mg²⁺ ionconcentration can be altered by addition of these ions or by removal ofthese ions by using chelating agents, such as ethylenediamine-tetraacetic acid (EDTA) (e.g., 5 mM), or ethyleneglycol-bis(β-aminoethyl ether)N,N,N′N′-Tetraacetic Acid (EGTA) (e.g., 5mM). Altering the free Ca²⁺ and Mg²⁺ ion concentration in the dilutedCSF-PKH has the effect of changing the extent of nuclear swelling andthe appearance of the chromatin within the nucleus. Very low or absentCa²⁺ and Mg²⁺ ion levels enhance nuclear swelling and chromatindecompaction. Increasing Ca²⁺ to 1.2 mM prevents significant nuclearswelling and chromatin decompaction. The optimal amount of Ca²⁺ andMg²⁺, and chelator can be empirically determined by varying the amountof chelator and cation concentration and measuring nuclei swelling.

[0166] Chelating agents or other agents which cause chromatindecondensation, such as polyanions like heparin and TPP or thiolreducing agents like DTT, need not be added directly to the dilutedCSF-PKH extract. These additional agents may be used to further swell ordecondense the nuclei after treatment in diluted CSF-PKH extract.

III. USE OF A CSF EXTRACT SUPPLEMENTED WITH A CYCLIN

[0167] A CSF extract supplemented with a cyclin can be used to inducechromosome formation without DNA replication. The CSF extract should besupplemented with a cyclin such as cyclin-

90 in an amount sufficient to achieve both nuclear envelope breakdownand nuclear chromosome formation. The cyclin is expected to act byraising the level of MPF activity in CSF extracts, and thereby, induceconversion of isolated nuclei into mitotic chromosomes.

[0168] CSF extract supplemented with cyclin was used to activate nucleiin suspension resulting in nuclei conversion into chromosomes. However,the activation also resulted in intermingling of the formed chromosome,and the chromosomes were stretched and sheared. The use of CSF extractsupplemented with cyclin-

90 may be improved by sticking trypsin treated nuclei to a glass surfaceprior to incubating with modified CSF.

IV. ACTIVATION OF MAMMALIAN SPERM

[0169] The present invention also features a method for activatingmammalian sperm, which is particularly suitable for the activation ofhuman sperm. The method involves the pretreatment of human sperm with aprotease, then activating the sperm with an activating egg extract. Thepresent disclosure is believed to be the first describing the use oftrypsin and an activating egg extract to activate a human sperm. Thepretreated sperm can be activated using activating egg extract, or thevarious procedures described herein, such as using a modified CSFextract (e.g., supplemented with an aqueous solution and/or a proteinkinase inhibitor, or supplemented with a cyclin) to achieve nuclearswelling or chromosome formation, and using both a CSF extract andactivating extract to cause nuclear activation.

[0170] The preferred method for activating a sperm involves (1)pretreatment involving a protease, a detergent, a thiol reducing agent,and preferably a thiol blocking to prevent reassociation of sulfhydrylgroups; (2) further pretreatment using CSF extract; and (3) activationusing an activation extract. Sperm can be obtained using techniquesknown in the art. Pretreatment can be carried out using a protease and adetergent either sequentially, or at the same time, followed by a thiolreducing agent, followed by a thiol blocking agent.

[0171] An example of a preferred protocol is as follows:

[0172] 1. Lyse sperm in 100 μg/ml lysolecithin for 5 min at 25° C.

[0173] 2. Treat with 100 μg/ml trypsin for 5-15 minutes (10 minutes isoptimum) at 25° C.

[0174] 3. Stop the lysolecithin and trypsin treatment by using 30 μg/mlsoybean trypsin inhibitor and 0.4% bovine serum albumin.

[0175] 4. Incubate sperm nuclei in 5 mM dithiothreitol for 60 minutes at4° C.

[0176] 5. Stop reaction 4 by incubating nuclei in 1 mM N-ethylmaleimidefor 10 minutes at 25° C.

[0177] 6. Incubate nuclei in CSF extract for 90 min at 25° C., followedby 60 minutes at 4° C.

[0178] 7. Incubate nuclei in activating egg extract.

[0179] The above procedure results in nuclear swelling without nuclearenvelope formation during the CSF pretreatment step (step 6), andadditional swelling, nuclear envelope formation, and DNA replicationduring the activating egg extract step (step 7). Steps 1-3, or theirequivalent, are all required to achieve complete swelling, nuclearenvelope formation, and DNA replication.

[0180] Activation of sperm cells have various uses including being usedto determine whether the sperm contains a particular gene or nucleicacid sequence which can be passed on during fertilization. Such studiesare useful, for example, to study the effect of aging on sperm; detectchromosomal defects; and determine whether foreign genes (such as thosepresent in the human immuno deficiency virus (HIV)) are present issperm.

[0181] An example of the usefulness of this aspect of the invention isin the field of animal breeding, particularly the breeding oftransgenically modified animals. Transgenically modified animals areusually created by injecting DNA sequences into early embryos. If theinjected DNA integrates into the host cell genome, it may end up in thegerm line of the adult animal after the animal matures. Transgenic maleanimals are particularly desirable since they can be bred to manyfemales. However, prior to breeding the percentage of modified germcells, as well as the copy number and distribution of the inserted genesin each cell is not known.

[0182] The methods and reagents provided herein for activating spermcell nuclei and examining their genetic composition, for example via insitu hybridization, make it possible to determine the percentage ofsperm carrying one or more copies of the inserted gene. This informationcan used to access the likelihood that a particular gene will be passedon to a future generation, prior to breeding the animal. Suchinformation is desirable because of the time and expense required tobreed an animal.

V. NUCLEAR ACTIVATION ASSAY

[0183] The procedures disclosed by the present invention, to activatenuclei, can also be used as a general assay procedure to measure nuclearactivation and the presence of a nucleic acid sequence in activatednuclei. The assay would be particularly useful to identify and purifyfactors present in CSF extract and to study male fertility.

[0184] A basic assay to measure DNA replication of activated cell couldhave the following steps: isolating a nucleus, pretreating the nucleus,further pretreating the nucleus, contacting the further pretreatednucleus with activating egg extract containing labeled nucleotides, anddetecting incorporation of label into replicated DNA. Preferably, aradioactive nucleotide would be used to determine activation bymeasuring the extent of label incorporated into newly synthesized DNA.

[0185] The assay could be tailored to aid in the purification of factorspresent in CSF which help prepare nuclei for subsequent activation.Specifically, the assay would be performed without the addition of CSFextracts. Rather, various fractions of CSF extract would be obtained bystandard purification techniques, and used instead of CSF extract. Thosefractions which increase activation activity can then be furtherpurified.

[0186] Another use of a nuclear activation assay is to study malefertility by measuring the extent of activation of human sperm underdifferent conditions. Such studies can be used, for example, to examinetechniques to preserve sperm so the sperm can be later used in in vitrofertilization, to test sperm of infertile men to identify causes of maleinfertility (see, Brown et al., Yale Journal Of Biology And Medicine65:29 (1992) (not admitted to be prior art), and test possible malecontraceptives.

[0187] In virtually all species of animals, sperm cells undergo tworeactions, capacitation and the acrosome response, before reaching andfusing with the egg surface. After the sperm nucleus enters an egg itundergoes several changes. The nucleus swells, acquires a nuclearenvelope and lamina, replicates its DNA, and eventually fuses with thefemale pronucleus. During this process, sperm basic proteins (histonesand protamines), are exchanged for embryonic histones.

[0188] It appears that in order for a sperm nucleus to respond to an eggcytoplasm it must first undergo some form of proteolytic digestion. Alikely site of necessary proteolytic digestion are non-histonecytoskeletal proteins. Possible contraceptives could target necessaryproteolytic enzymes. The affect of the contraceptive could be determinedby assaying the degree to which activation is inhibited. Possiblecontraceptives could also target other enzymes which may be needed foractivation.

[0189] Alternatively, the assay could be used to determine conditionswhich result in higher levels of activation thereby finding conditionswhich enhance fertilization.

[0190] Specific uses of the nuclear activation assay include thefollowing:

[0191] 1) Assaying sperm cell treated under different conditions ofpreparation, cryopreservation, capacitation and handling;

[0192] 2) Assaying the affect of sperm cell enzymes (e.g., proteases,nucleases, phosphatases, and kinases), including the inhibition of spermcell enzymes, on activation;

[0193] 3) An assay to purify enzymes affecting activation;

[0194] 4) Assaying the sperm from infertile individuals to determine ifinfertility is due to problems with sperm nuclear activation;

[0195] 5) Assaying the ability of specific drugs or reagents to enhanceor inhibit activation;

[0196] 6) Assaying the affect of inhibitors or activators of sperm cellenzymes on activation;

[0197] 7) Assaying for the presence of a gene used to create atransgenic animal; and

[0198] 8) Assaying for the presence of viral genome, such as HIV presentin sperm.

[0199] The specific nuclear activation assay used to study fertilitywould be tailored to study a particular aspect of activation. Forexample, to assay the effect of reagents on activation the sperm shouldbe handled and prepared under mild conditions. As discussed above mildconditions are useful in minimizing inadvertent activation of proteasesor nucleases. To obtain sperm for the activation assay, fresh spermsamples should be first washed in isotonic saline solution under mildconditions. The sperm can then be stored by freezing in liquid nitrogenunder controlled conditions in the presence of a cryoprotectant. Martinet al., in PREIMPLANTATION GENETICS, Plenum Press, New York (Verinskyand Kuliev, eds, 1991).

[0200] Fresh or frozen/thawed sperm can be treated under conditionswhich result in capacitation as described by Martin et al. Supra. Thesperm membrane is then permeabilized under mild conditions as describedabove (e.g., lysolecithin is used to permeabilize the membrane). Thenuclei are then recovered from lysed sperm by mild centrifugation inisosmotic buffer. Nuclei are preferably pretreated as described above inSection II (e.g., using a membrane permeabilizer, a protease, and athiol reducing agent).

[0201] The nuclei can then be further pretreated (e.g., using CSFextract containing 100 μM Ca²⁺). The further pretreated nuclei arecontacted with activating egg extract and activation activity ismeasured using standard techniques such as using a labeled reagent(e.g., P³²-CTP) to detect DNA replication, and microscopic visualizationof nuclear swelling. Additionally, in situ hybridization can be carriedout to determine the presence, number and location of particular DNAsequences. The effect of various reagents on nuclear activation can bedetermined by adding reagents to the sperm before or after the variousindividual steps of isolation, pretreatment, further pretreatment orcontact with activating egg extract.

VI. RETROVIRAL INTEGRATION ASSAY

[0202] The assays described herein can be used to examine integration ofproviral nucleic acid, such as from HIV, into host DNA. The assays canbe carried out using a whole nucleus or a pseudonucleus. Such assays canbe used to identify target sites to inhibit proviral integration, and toderive anti-viral agents directed at such target sites.

[0203] A provirus is the double-stranded DNA form of a retrovirus. It issynthesized in the cytoplasm of a cell infected with a retrovirus byreverse transcription of the viral RNA. Integration of the provirus DNAinto the host cell genome is a critical step in the life cycle of allretroviruses, including HIV-1, and leads to viral expression and newvirus production. Thus, by blocking viral integration, viral propagation(e.g., viral multiplication and/or viral infection) can be inhibited.

[0204] An integration assay can be performed as follows:

[0205] 1. A cell nucleus is pretreated to separate the nucleus from itssurrounding cytoskeleton to form a pretreated nucleus. The choice ofcell nucleus can be varied depending on the virus studied. Preferably,the cell nucleus will be obtained from a cell which is a natural hostfor the virus. Examples of cells susceptible to retroviral infectioninclude mammal and plant cells. Preferably, a human cell nucleus isused.

[0206] 2. The pretreated nucleus is activated and incubated with a viralintegration complex. A viral integration complex contains the proviraldouble stranded DNA form of the viral RNA and material needed for viralintegration. Thus, the integration complex contains an integrase and maycontain other viral enzymes and proteins. An integration complex can beobtained by one skilled in the art using standard techniques. Forexample, a high speed supernatant of cells infected with a virus can beused as an integration complex. (Brown, et al., Cell 49:347, 1987).Alternatively, an integration complex can be obtained from purifiedviral integrase and specific oligonucleotides having the viral sequencesneeded for integration (Engelman et al., Cell 67:1211, 1991). Theintegration complex can be added to nuclei, chromatin or pseudonuclei,at different points in the cell cycle. For example, incubation can takeplace before (e.g., prior to activation), during the time that nucleiare swelling and forming nuclear envelopes in CSF extract and activatingextract, or during chromatin assembly, nuclear envelope formation orreplication of pseudonuclei.

[0207] 3. Measuring integration of the viral nucleic acid into the hostnucleic acid. The measurement can be carried out using standardtechniques such as through the use of hybridization probes targeted toviral nucleic acid sequences. The use of hybridization probes can befacilitated by amplification techniques such as PCR amplification.Preferably, unintegrated viral nucleic acid is separated from hostnuclei acid prior to using the hybridization assay probe. A separationstep is useful for decreasing hybridization of probes to viral nucleicacid not incorporated into the host genome. Separation can be carriedout, for example, by centrifugation of nuclei through glycerol orelectrophoresis of isolated nucleic acids.

[0208] Alternatively an integration assay can be carried out using apseudonucleus. A pseudonucleus can be constructed from a plasmid DNAtemplate which is then used as a target for retroviral integrationrather than intact activated nucleus. This approach has the advantagethat the oligonucleotide size of the plasmid genome is much smaller thanthat of a whole eukaryotic nucleus and the sequence of the plasmidgenome is either known or can be readily established.

[0209] A pseudonucleus can be constructed by adding plasmid DNA to afresh or frozen/thawed CSF extract (e.g., at 0.1-20 ng/μl). Thismaterial can be used immediately or frozen for latter use. The plasmidDNA can form chromatin in the CSF extract. (For example, see Sanchez etal., Journal of Cell Science 103:907, 1992 and Wangh, Journal of CellScience 93:1, 1989, describing such chromatin formation using plasmidFV1 dervived from type 1 BPV in intact Xenopus eggs).

[0210] The assay is carried out by activating the chromatin andmeasuring integration of viral nucleic acid. For example, the chromatinis diluted into an additional sample of CSF extract which is activated(e.g., by the addition of 1.2 mM Ca²⁺). Activation triggers nuclearenvelope formation around the chromatin and causes the chromatin toreplicate.

[0211] Thus, an activation assay can be performed using a pseudonucleusin place of a pretreated nucleus as follows: 1) forming a pseudonucleus;2) activating the pseudonucleus, and incubating with an integrationcomplex containing viral nucleic acid before activation or at differenttimes after activation; and 3) measuring integration of the viralnucleic acid into the nucleic acid of the pseudonucleus.

[0212] Using an integration assay, it can be determined when viralintegration occurs during the cell cycle, and if an agent is effectivein inhibiting viral integration. For example, the importance ofdifferent stages of the cell cycle in viral integration can be evaluatedusing CSF or activating extracts supplemented with DMAP, aphidicolin,and inhibitors of type II topoisomerase. DMAP and aphidicolin block DNAreplication but allow nuclear swelling and chromatin decondensation toproceed. Inhibitors of type II topoisomerase block chromatindecondensation, which requires type II topoisomerase activity. Examplesof the use of such drugs include the following:

[0213] 1) If drugs such as DMAP and aphidicolin inhibit chromatindecondensation but fail to inhibit viral integration, then chromatindecondensation after mitosis is probably all that is necessary forintegration. In this instance, drugs could be designed to preventintegration during or prior to chromatin decondensation.

[0214] 2) If integration (i.e., insertion of the proviral DNA into thetarget genome) and circularization (i.e., insertion of the proviral DNAinto itself) are both blocked by DMAP and aphidicolin, then on-going DNAsynthesis is probably required for viral integration. Accordingly viralintegration during DNA synthesis could be targeted. If DNA synthesis isrequired for proviral integration, it can then be determined whetherintegration occurs before or after the host genome target is replicated.For example, bromodeoxyuridine triphosphate (BrdUTP) can be added toreactions to increase the density of newly synthesized DNA strands.Samples can then be collected at the end of the S phase when genomereplication is complete. After electrophoretically removing allunintegrated viral molecules, the genomic DNA can then be cleaved with arestriction enzyme that recognizes two or more sites within the viralgenome. The preparation can then be fractionated, for example, by CsCldensity gradient centrifugation and probed for released segments of thevirus. If the provirus is inserted into the host genome beforereplication, the viral DNA will be recovered in the heavy/light densitypeak, along with virtually all the genomic DNA. On the other hand, ifthe provirus is inserted after its target sequence has replicated, theviral DNA will be in the light/light peak. After determining the timingof integration, drugs can be designed to inhibit integration and tested.

[0215] 3) If integration takes place in the presence of DMAP oraphidicolin, but not both, this would indicate that DNA synthesis, perse, is not required for integration, but cell cycle-dependent propertiesof the cytoplasm influences integration.

[0216] Thus, using this application as a guide, one skilled in the artcan identify when, during the life cycle of a cell, viral integrationoccurs, target drugs to inhibit such viral integration, and assaywhether an agent inhibits viral integration. Agents which inhibitretroviral integration may be used as therapeutic agents to treat aperson infected with a retrovirus (such as HIV) or prevent an uninfectedperson from being infected with a retrovirus. A retroviral “therapeuticagent” refers to an agent which reduces, to some extent, the in vivopropagation of a retrovirus and preferably reduces, to some extent, oneor more of the symptoms associated with a retroviral infection.

VII. MICROCHAMBER MICROSCOPE SLIDE

[0217] Conversion and analysis of interphase nuclei to meiotic ormitotic chromosomes is facilitated using the microchamber microscopeslide. Referring to FIG. 4, there is shown a microchamber microscopeslide 10. The microchamber microscope slide allows very small amounts ofexpensive and hard to come by reagents to be used sequentially on nucleiin situ. For instance, isolated nuclei can be placed into centralmicrochamber 20 which is formed tear-drop shaped, pretreated, swelled,converted to chromosomes, stained, and then read or analyzed withoutfurther centrifugation or complex manipulation.

[0218] A thin strip of PARAFILM® wax 12, or other appropriate waterresistant plastic tape or like material, is annealed to a standardmicroscope slide 11 or coverslip 14. The microscope slide 11 isgenerally flat and rectangular-shaped with a top and bottom side. Thecoverslip 14 is generally flat and circular-shaped with a top and bottomside. The PARAFILM® strip defines three wells connected by two narrowchannels. Center micro-chamber 20 is generally teardrop-shaped with agenerally rounded head end and a generally arrow shaped tail end. Thevolume of the microchamber is preferably between 5 μl and 50 μl, mostideally between 10 μl and 20 μl.

[0219] The head end of microchamber 20 is connected to a fill well 16 bya narrow entrance channel 18. The tail end of microchamber 20 isconnected to a drain well 24 by a narrow exit channel 22. The volume ofthe resulting wells is determined by the thickness of PARAFILM strip 12and the size and shape of the wells; these parameters are adjustable.

[0220] Microchamber 20 is covered by a thin inverted coverslip 14. Athin inverted coverslip is best suited for use with an upright compoundmicroscope. Other types of coverslips may be used. For example, anoptically thin coverslip is suited for use with an inverted compoundmicroscope.

[0221] In the preferred mode of operation, coverslip 14 completelycovers microchamber 20 leaving fill well 16 and drain well 24substantially uncovered. A sample of cells, nuclei, or other material,is pipetted to the wide part of the microchamber. The microchamber isthen covered with a coverslip which is caused to adhere to the uppersurface of the PARAFILM® strip 12 by applying two small drops ofparaffin oil. The overlaying coverslip can be siliconized to minimizesticking of water and other materials to this surface.

[0222] Microchamber 20 is filled by capillary action by placing fluid infill well 16. Excess fluid is then removed from both the fill well anddrain well 24. The microchamber is flushed by placing fluid in the fillwell and then sucking the fluid through the microchamber by capillaryaction achieved by touching blotting paper to the edge of the drainwell.

[0223] The general teardrop shape enhances flushing of microchamber 20.For a 10 μl microchamber as little as 20 μl of fluid is sufficient toclear the microchamber. If necessary, the coverslip 14 can be removedand the fill channel 18 and exit channel 22 sealed with a small bead ofsilicone stopcock grease. Material can then be recovered from themicrochamber.

[0224] The microchamber microscope slide is extremely versatile. It canbe sterilized, placed in tissue culture medium, and used as a growingsurface for cells. Further, it is possible to increase the depth of thetwo side wells while leaving the microchamber shallow. Each of the sidewells could be covered with their own lid. One of the side wells couldbe filled several millimeters deep with tissue culture medium while theother well is left unfilled. Tissue culture medium would then flowthrough the microchamber across the cells until the two side wells reachequilibrium, the exact flow rate being adjustable. Further potentialuses include the following: (1) analysis of growing cells; (2) analysisof isolated cells, particularly fetal blood cells; (3) analysis of cellnuclei, or other subcellular particles, organelles or materials; and (4)analysis of material of non-living origin.

[0225] The microchamber microscope slide allows analysis of material byessentially all light microscopy staining techniques including thefollowing: (1) fluorescent microscopy of incorporated precursors,antibody staining, nucleic acid hybridization techniques; (2)conventional histological staining procedures; and (3) staining based onenzymatic amplification of molecular signals.

[0226] The microchamber microscope slide also allows analysis ofbiological material by incorporation of radioactive precursors, followedby autoradiographic detection of the incorporated precursors.

[0227] The microchamber microscope slide also allows “on-line”microscopic observation of material being treated or altered by fluidsflowing through the microchamber. In particular, the microchambermicroscope slide is ideally used to both isolate and analyze fetal cellfrom maternal blood. Isolation may be achieved by first coating themicrochamber with the appropriate antibody to fetal cells, or theiralready isolated nuclei, and the microchamber is otherwise not sticky.The microchamber itself selects and holds the fetal cells, or nuclei,while the maternal cells, or nuclei, are washed away. The fetal cells,or nuclei, can then be fluorescently tagged in situ and their positionsidentified even before starting in vitro nuclear swelling and chromosomeformation. The fetal cells or nuclei, can then be activated using theappropriate treatments described herein.

VIII. ACTIVATION KITS

[0228] The technology disclosed in the present invention can be used toproduce activation kits useful for clinical activation of nuclei andscientific research. These kits are particularly useful for prenatalscreening. Uses of an activation kit to aid in scientific researchinclude facilitating the study of complex biochemical activitiesincluding the assembly of nucleosomes and chromatin on plasmid or viralDNA, formation of eukaryotic nuclear envelopes surrounding nucleartemplates, semi-conservative replication of double stranded DNA withineukaryotic nuclei, conservative repair replication of single strandedDNA independent of nuclear envelope assembly, activation of quiescentcell nuclei, nuclear envelope breakdown, condensation of chromatin intochromosomes, formation of meiotic and mitotic spindles, regulatedtranscription of eukaryotic genes, and protein synthesis.

[0229] A basic activation kit comprises frozen activating egg extractand frozen CSF extract. These extracts are prepared based upon themethods described in the present invention. Preferably the kit containsfrozen activating egg prepared from eggs having an elevated DNAsynthesis activation activity. More advanced kits contain varioussupplements which aid in activation. The various supplements are eitherin separate containers present in the frozen activating egg extract orfrozen CSF extract.

[0230] Preferably, these supplements are in separate containers. Usefulsupplements includes CaCl₂, nocodazole, β-glycerol-PO₄,phosphodiesterase inhibitor (e.g., caffeine), cAMP, protein kinaseinhibitor (e.g., DMAP). Preferably, the activation kit contains amicrochamber microscope slide.

[0231] The activation kits could also be supplemented with reagents usedto study activation in general or determine the extent of genomereplication. Useful supplements for these activities include radioactivenucleotides, biotinylated nucleotides and different dyes (e.g.,biotin-Texas Red streptavidin and Hoechst).

IX. CLONING WHOLE ANIMALS FROM SOMATIC CELL NUCLEI

[0232] The procedures disclosed by the present invention, to activatenuclei, are also useful for preparing a nucleus for subsequenttransplantation into an egg for the purpose of directing the developmentof a new organism. Prior to nuclear transplantation, the nucleus to betransplanted is activated in vitro. The activated nucleus is thentransplanted into an egg whose own nucleus has either been removed orfunctionally inactivated. The egg subsequently develops into an neworganism under the direction of genetic information contained in thetransplanted nucleus. Uses of cloning somatic cell nuclei include,creation of a clone of genetically identical animals, cloning animalshaving favorable attributes, and producing more animals which are indanger of becoming extinct.

[0233] A difficulty in cloning somatic cell nuclei from mammalianspecies is that these nuclei are imprinted with patterns of genestructure and function (e.g., DNA methylation patterns) which differfrom sperm and egg nuclei patterns. Thus, it is necessary to reprogramsomatic cell nuclei before cloning to eliminate the different patterns.Prior activation of somatic cell nuclei in an appropriate egg extractbefore transplanting should allow for the necessary reprogramming toenable a transplanted nucleus to give rise to either a complete, orsubstantially complete new organism.

[0234] Cloning using a somatic cell nucleus comprises three steps; (1)activating the somatic cell nucleus, (2) preparing a recipient egg, and(3) transplanting the somatic cell nucleus into the egg. The first stepis preferably carried out using the improved procedures, disclosedabove, to activate a nucleus. Preferably isolation, pretreatment,further pretreatment, and contact with activating egg extract arepreformed under conditions where the activated nucleus has a high DNAsynthesis activation activity.

[0235] Preparation of a recipient egg will vary depending upon the eggsource. The egg source should be treated in a manner to preventactivation before nuclear transplantation. Procedures to preparemammalian eggs, such as those described by Martin et al. supra, are knowin the art.

[0236] Preparation of a recipient egg includes destroying the egg'spronucleus. Destruction or removal of the egg's own nucleus guaranteesthat the eggs genetic material (DNA) does not contribute to the growthand development of the newly cloned individual. One method of destroyingthe pronucleus is by using ultraviolet light as described by Gurdon, inMETHODS IN CELL BIOLOGY. XENOPUS LAEVIS:PRACTICAL USES IN CELL ANDMOLECULAR BIOLOGY, 36:299-309, Academic Press, California. (Kay and Pengeds., 1991). Alternatively, the egg pronucleus can be surgically removedby procedures known in the art such as those described by King, inMETHODS IN CELL PHYSIOLOGY 2:1-36, Academic Press, New York (D. M.Prescott, ed., 1966), and McGrath and Solter, Science 220:1300-1319(1983).

[0237] Nuclear transplantation can be carried out by standardtechniques. These techniques, vary depending upon the species, and areknown in the art.

[0238] It should be possible to clone Xenopus in the following manner:nuclei from Xenopus red blood cells are isolated, pretreated, andfurther pretreated. Nuclei are then activated by contact with anactivating egg extract. The nuclei are then activated to differentstages in the cell cycle (e.g., S-phase, G2, etc.), and transferred torecipient prepared Xenopus eggs.

[0239] Recipient Xenopus eggs are prepared for nuclear transplantationby hardening using Ca²⁺ (as described above), and then irradiating withultraviolet light to destroy the egg's genome. One to two activatedsomatic nuclei, in 20 to 50 nanoliters are then microinjected into theXenopus egg, into the clear cytoplasmic region that lies approximately400 microns below the animal pole of the egg. The egg is then incubatedunder conditions that permit cytoplasm rotation. These conditions can beconveniently obtained by floating the egg on Metrizamide®. Rotation ofthe egg cytoplasm relative to the egg cortex is important forestablishment of the proper dorsal/ventral axis of the developingvertebrate embryo.

X. EXAMPLES Example 1 Further Induction Optimization

[0240] This example describes additional experiments carried out tofurther determine the optimal induction time for an activating eggextract. Protein synthesis during the early part of the first cell cyclein activated eggs or egg extracts is required for preparation of anactivating extract capable of efficient and complete genome replication.The required proteins can either be synthesized in intact eggs beforepreparation of extracts or in extracts including frozen/thawed extracts.As noted above, activating egg extract should be prepared from extractsinduced for more than 10 minutes to enhance DNA synthesis activationactivity.

[0241] It was found that proteins synthesized during the first 28-30minutes in intact eggs (incubated at 20° C.) or during the first 60-80minutes in a freshly prepared and activated extract (incubated at 25°C.), promote subsequent DNA replication. In contrast, proteinssynthesized later in the first cell cycle, i.e., after replication isunderway, inhibit DNA synthesis. The changes in DNA synthesis can bedetected as alterations in the time which DNA synthesis starts, theinitial rate of replication, and the overall amount of replication.

[0242] The amount of Cacl₂ used to induce a freshly prepared CSF extractregulates whether or not the extract exits meiotic metaphase arrest,traverses the first interphase, and re-enters the first M-phase. Asjudged by measurements of histone H1 kinase activity, fresh CSF extractinduced by the addition of 3 mM CaCl₂ exits meiotic metaphase, entersinterphase, but fails to enter mitosis-I. In contrast, CSF extractinduced with 1.2 mM CaCl₂ exits meiotic metaphase, enters interphase,and then proceeds into mitosis-I, as indicated by a second peak in H1kinase activity.

[0243] For the purpose of comparison, extracts were prepared from eggsinduced and incubated at 20° C. for varying lengths of time before beingcrushed. In all cases the eggs were amassed, induced, washed, crushed,and extracts were prepared as described for prepared activating extractswith the following modifications: (1) all tubes and pipette tips used toprepare egg extracts were first treated with 1% diethylpyrocarbonate todestroy ribonuclease activity and (2) all the steps in extractpreparation were carried out using plastic gloves to avoid ribonucleasecontamination. Extracts were frozen on an aluminum block, chilled withliquid nitrogen. and then thawed at a later time prior to being used.DNA synthesis was followed by incorporation of P³²dCTP, followed byelectrophoresis and phosphoimager analysis.

[0244] The results demonstrate that optimal DNA synthesis activatingextracts are obtained by synchronously inducing batches of eggs andincubating them for 28-30 minutes at 20° C. Of the time periods testedthe 28-30 minute extracts initiated nuclear replication earliest,synthesized DNA fastest, and replicated more DNA, then egg extractsinduced for 10 minutes, 22 minutes, 34 minutes or 40 minutes. Theoverall order for earlier nuclear replication, faster DNA synthesis, andextent of DNA replication was as follows: 10 minutes<22 minutes<25minutes<28 minutes>34 minutes>40 minutes. Because the cell cycle of theegg is so rapid, even small differences in the length of incubationperiod or the temperature of incubation result in suboptimal extracts.

[0245] It was also determined that maximal replication, even in thefrozen/thawed 28-30 minute extract, depends on continuing proteinsynthesis during the first 30 minutes of the in vitro incubation.Activating egg extract were prepared as described above, induced for 28minutes. Cycloheximide was added just prior to induction, or 30 minutesafter induction. Maximal DNA replication was observed for control (nocycloheximide) and cycloheximide added 30 minutes after induction, whilezero minute cycloheximide addition resulted in significantly less DNAreplication. This suggests that the proteins required for efficientreplication are relatively unstable but are abundantly synthesized frommRNAs recruited onto polysomes during the first 28-30 minutes followingegg induction.

Example 2 cAMP Supplemented Activating Egg Extract

[0246] The affect of cAMP on DNA replication in activated Xenopus redblood cells was determined. Xenopus nuclei were isolated and pretreatedby a method based on Coppock et al., Developmental Biology 131:102(1989), as follows: Xenopus blood was obtained from females by cardiacpuncture and collected using a syringe half-filled with Barth's solution(88 mM NaCl, 2.3 mM KCl, 0.82 mM MgCl₂ and 10 mM Hepes, pH 7.4)containing heparin (10 mg/ml); the blood was immediately diluted into 10ml of ice-cold 0.6×SSC (1×SSC is 0.15 M NaCl, 0.015 M Sodium citrate, pH7.0) containing 0.1 mg/ml heparin, 0.1 mM TPCK (N-tosyl-L-phenylalaninechloromethyl ketone), 0.1 mM TLCK (Na-p-Tosyl-L-lysine chloromethylketone), 0.05 mM PMSF (phenylmethylsulfonyl fluoride), 5 μg/mlleupeptin, and 31.25 mM Na₂S₂O₅; bleeds containing clots, even smallones, were rejected; diluted blood was underlaid with 0.5 volumes of icecold Metrizamide® (refractive index of 1.3660 in 0.6×SSC) andcentrifuged at 180 g for 10 minutes at 4° C., red blood cells pelletedbelow Metrizamide® while white cells banded above Metrizamide®; the redcell pellet was resuspended using 0.6×SSC and centrifuged inMetrizamide® four more times to obtain erythrocytes of greater than99.9% purity; cells were washed three times in NIB and resuspended at2×10⁸ cells/ml; cells were then resuspended in NIB:glycerol (7:3) andfrozen in aliquots of 100 μl in liquid nitrogen; before using, frozencells were thawed, diluted to 4×10⁷ cells/ml in NIB at 23° C., and addedto an equal volume of NIB containing 80 μg/ml lysolecithin (40 μg/mlfinal concentration) and 0.6 μg/ml trypsin (0.3 μg/ml finalconcentration) (the trypsin used in this example, and the other examplesdescribed herein, was Sigma brand Type XIII trypsin, TPCK treated fromBovine Pancrease, approximately 11,000 units/mg solid); after 5 minuteslysolecithin and protease treatment was stopped by adding soybeantrypsin inhibitor to a concentration of 30 μg/ml and bovine serumalbumin to a final concentration of 0.4%; the resulting nuclei werecentrifuged at 800 g at 0° C. for 10 minutes, washed twice in NIB,resuspended with ice-cold NIB and kept on ice.

[0247] Isolated and pretreated nuclei were added at 200 nuclei/μl to 550μl thawed “prepared activating egg extract” supplemented with 5 μg/mlnocodazole, 250 μg/ml cycloheximide, 10 μCi P³²-dCTP, 10 mM creatinephosphate, and 10 μg/ml creatine phosphokinase. Cyclic-AMP was thenadded to separate aliquots to yield final concentrations of 0.0 μM, 0.1μM, 1.0 μM, or 10 μM.

[0248] Each aliquot was warmed to 23° C. and sampled over time todetermine P³²-dCTP incorporation into replicated DNA. At each timepoint, a 7 μl aliquot was taken, frozen on dry ice, and later thawed anddigested by the addition of 10 μl replication sample buffer (80 mM Tris(pH 8.0), 8 MM EGTA, 0.13% phosphoric acid, 10% Ficoll, 5% SDS, 0.2%bromphenol blue) containing proteinase K (1.0 mg/ml) for 2 hours at roomtemperature. Incorporated radioactivity was analyzed by electrophoresison a 0.8% agarose gel (50V, 20 hours) followed by vacuum drying the geland counting on a Betascope.

[0249] As indicated by Table 2, the addition of 10 μM cAMP inhibits DNAreplication in activated nuclei as compared to DNA replication occurringwithout any cAMP. DNA replication increased with 0.1 μM and 1.0 μM cAMP.A greater increase was seen with 1.0 μM than with 0.1 μM cAMP. Thus,cAMP can be used to increase the activation activity of activating eggextracts. A concentration of approximately 0.3 μM, was used insubsequent studies. TABLE 2 Micromoles Cpm Incorporated of Cyclic AMPAdded After X Minutes 0.0 0.1 1.0 10 0 14 14 10 43 45 37 51 14 34 90 7368 56 31 135 288 290 564 35 180 839 1,141 2,316 85 240 1,556 2,945 4,484168 300 2,954 2,692 5,504 571

Example 3 Caffeine Supplemented Activating Egg Extract

[0250] The effect of caffeine on DNA replication in activated Xenopusred blood cells was determined. The experimental conditions used were asdescribed in Example 1 with the following changes: the concentration ofcAMP was set at 0.3 μM and caffeine was added to the activating eggextract to a concentration of either 0.2 mM, 1.0 mM, or 5.0 mM.

[0251] As illustrated by FIG. 1, caffeine at 1.0 mM in the presence of0.3 μM cAMP gave the highest initial rate and extent of DNA replicationin activated nuclei. Thus, caffeine can increase the activation activityof an activating egg extract.

Example 4 CSF Extract Supplemented With DMAP and Treated With ActivatingExtract

[0252] Addition of 6-dimethylaminopurine (DMAP) to CSF extracts was usedto further pretreat Xenopus erythrocyte nuclei and stimulate subsequentDNA replication in activating egg extract. DMAP can be used to inhibitnucleic acid synthesis and protein kinase activity. Xenopus erythrocytenuclei were isolated and pretreated as described in Example 1 above, andincubated in thawed “prepared CSF extract” supplemented with 80 mMβ-glycerol-PO₄, and 5 μg/ml nocodazole at a concentration of 2000nuclei/μl. Further pretreatment was carried out by incubation for 30minutes at 4° C., then 30 minutes at 25° C., then 60 minutes at 4° C.Half the samples were supplemented with 5 mM DMAP before addition of thenuclei. After the two hours of incubation, each sample was diluted with9 volumes of activating egg extract, supplemented with 5 μg/mlnocodazole (this dose of nocodazole slows down the rate of replication)and approximately 160 μCi/ml P³²-dCTP. Aliquots were removed over timeto measure DNA replication.

[0253] As illustrated by FIG. 2, the addition of DMAP to CSF extractsenhanced the ability of the CSF extract to stimulate subsequent DNAreplication in activating egg extract. DMAP decreased the lag timebefore the onset of replication and increased the initial rate and totalamount of DNA synthesis.

Example 5 Warm-Then-Cold Regime

[0254] Various warm-then-cold regimes used as part of a furtherpretreatment increased DNA replication in activated nuclei. ThawedXenopus erythrocyte nuclei (isolated and pretreated as in Example 1above) were added at 2000 nuclei/μl to thawed “prepared CSF extract,”supplemented with 80 mM β-glycerol-PO₄. The mixture was incubated usingvarious warm-then-cold regimes. At the end of each incubation periodsamples was diluted with 9 volumes of “prepared activating egg extract”supplemented with 5 μg/ml nocodazole and P³²-dCTP. Samples were removedover time to measure the extent of DNA replication.

[0255] As shown by the data represented in FIG. 3, the followingwarm-then-cold regimes stimulated subsequent DNA replication: 30 minutesat 25° C., and 90 minutes at 4° C.; 30 minutes at 4° C., 30-minutes at25° C., and 60 minutes at 4° C.; 60 minutes at 4° C., 30 minutes at 25°C., and 30 minutes at 4° C. Incubation for 90 minutes at 4° C., and 30minutes at 25° C. was not as effective as incubation regimes that had awarm period followed by a cold period. Thus, nuclei activation ispreferably performed using a warm-then-cold regime.

Example 6 Activation Using Frozen/Thawed Extracts

[0256] Activation of Xenopus red blood cell nuclei was studied usingfrozen/thawed CSF extracts and frozen/thawed activating egg extract.Xenopus red blood cell nuclei were isolated and pretreated as describedin example 1. These nuclei were further pretreated at 2000 nuclei/μl inthawed “prepared CSF extract” supplemented with 10 mM creatinephosphate, 10 μg/ml creatine phosphokinase, 5 μg/ml nocodazole, 80 mMβ-glycerol-PO₄, 100 μM CaCl₂ and incubated using a warm-then-cold formatof 60 minutes at 25° C. followed by 60 minutes at 4° C.

[0257] After further pretreatment, samples were diluted with 9 volumesof thawed “prepared activating egg extract” containing 10 mM creatinephosphate, 10 μg/ml creatine phosphokinase, and incubated with either:A) 200 μCi/ml P³²-dCTP; B) 16 μM biotin-11-dUTP, 16 μM MgCl₂; or C) noadditions.

[0258] At various time intervals an aliquot of each incubation wastreated as follows:

[0259] A) P³²-labelled samples were treated with sodium dodecyl sulfate(SDS), proteinase-K, and then analyzed on agarose gels to determine DNAreplication. Total incorporated radioactivity was measured using aMolecular Dynamics phosphoimager. The sizes of the radioactive moleculeswere observed and photographed on X-ray film.

[0260] B) Biotin labelled nucleic acid was used to visualize replicatednuclear DNA. Biotin labelled samples were fixed by mixing into thesamples approximately 40 volumes of freshly prepared 1.0 mM ethyleneglycol bis-(succinic acid N-hydroxysuccinimide ester) (EGS) andincubating at 37° C. for 30 minutes. Fixed nuclei were stored at 8° C.for 48 hours and then centrifuged onto glass coverslips (2000 rpm, at 4°C. for 15 minutes) through a 25% glycerol layer. The glycerol layer wasremoved and the samples were stained with Texas Red-Streptavidin (GibcoBRL, diluted 1:40 in PBS). Coverslips were then washed with bufferedsaline and stained with 1.0 μg/ml Hoechst 33258 stain (for total DNA).Each sample was examined and photographed at 60× using an Olympusoptical system. Using these conditions: 1) nuclear envelopes weredetected under phase optics as a dark line around the nucleus; 2) totalnuclear DNA was observed under fluorescent optics as Hoechst positive(blue) staining; and 3) newly replicated biotinylated DNA were detectedas Texas Red positive (red) staining.

[0261] C) Samples from incubate (C) were used to measure histone Hikinase activity during the course of the experiment.

[0262] As judged by both P³²-dCTP incorporation and biotinylated-dUTPincorporation, new DNA replication in erythrocyte nuclei was highlysynchronous and efficient. Replication began at 30-40 minutes ofincubation and was completed by 80-90 minutes of incubation. Noadditional DNA synthesis was observed between 90-140 minutes. After 140minutes DNA replication resumed. The initial rate of DNA synthesis inthis system using a frozen/thawed activating egg extract is onlyslightly slower than that using fresh activating egg extract.Furthermore, it appears that replication of the entire genome wasachieved.

[0263] As judged by nuclear morphology and staining, swelling wasobserved about 20 minutes after addition of activating egg extract(T=20). At T=20 no DNA replication was observed. DNA synthesis andbiotinylated-dUTP incorporation were both first observed at T=40.Nuclear swelling continued until T=80 at which time nuclear condensationand nuclear envelope breakdown began. Photographs of 10 or more nucleiat each time point revealed that virtually all nuclei in each samplewere activated at the same time and in the same manner. Hoechst stainingand biotin labelling revealed that nuclear DNA was first highlycompacted (T=0), became more diffuse during the period of swelling andreplication (T=20 to T=80), and then condensed into chromosome likestructures (T=100 to T=180). Nuclear envelope breakdown occurred atT=100 to T=120 minutes, but mitotic spindle formation was not observedin these samples. This was likely due to the presence of low levels ofnocodazole (0.5 μg/ml). At T=160 many of the nuclei appeared under phasecontrast to have nuclear envelopes suggesting they entered a secondinterphase. All nuclei at T=180 had distinct chromosome-like structuresindicating that they entered a second mitosis. DNA synthesis (P³²-dCTPincorporation) resumed between T=140 and T=160 and then stopped at T=160in accord with a second S-phase followed by a second mitosis.

[0264] Histone H1 kinase levels were low at the start of S-phase (T=40)and rose gradually thereafter. Correlations of DNA synthesis, nuclearmorphology, and H1 kinase levels suggested that a threshold level of H1kinase leading to nuclear envelope breakdown and first mitosis wasreached at T=100. H1 kinase levels continued to rise until T=180,despite the fact that DNA synthesis resumed at T=140-160.

[0265] First mitosis occurred relatively early (T=100 to T=120) and wasnot accompanied by DNA fragmentation. These observations are consistentwith the view that genome replication was complete in this experiment.Agarose gel analysis of the p³²-labelled DNA demonstrated that virtuallyall newly synthesized DNA was initially of very high molecular weight(HMW), some of this material was then converted to pieces of a ratheruniform moderate molecular weight (MMW). DNA pieces of the MMW size arenot degradation products and reflect a fundamental unit of DNA packagingin condensing chromosomes. MMW may be due to experimental interruptionof topoisomerase II dependent deconcatenation of replicated DNA loops.

[0266] In summary this experiment demonstrates, an in vitro system usingfrozen/thawed CSF extracts, and frozen/thawed activating egg extractsprepared from Xenopus eggs. In this system nuclei swell, acquire newenvelopes, and cycle through at least one complete S phase followed byone complete M phase. Only a limited amount of DNA synthesis takes placein a second S-phase. This system permits highly synchronous activationand cycling of quiescent cell nuclei, and is directly applicable to theactivation of non-dividing human cells such as fetal cells includingkeratinocytes, trophoblasts, erythrocytes and leukocytes, and spermnuclei.

Example 7 Comparison of High Speed Versus Low Speed CSF Extract

[0267] This example compares nuclei activation using a pretreatment ineither low speed or high speed “prepared CSF extract.” Activation wascarried out using frozen/thawed activation extract induced for 28minutes. Nuclei activation was assayed by measuring DNA replication andHistone H1 kinase activity.

[0268] Xenopus erythrocyte nuclei were prepared as described in Example2 and incubated in either a low speed prepared “CSF extract” or a highspeed “prepared CSF extract.” In both cases nuclei were then dilutedinto 9 volumes of a 28 minute induced activating egg extract. For eachincubate one set of samples were collected to measure histone H1 kinaseactivity, another set of samples containing P³²-dCTP was used to measureDNA synthesis. The nuclei incubated in the high speed CSF extractreplicated and progressed through the cell cycle more synchronously thanthose incubated in low speed CSF extract.

Example 8 Use of Diluted CSF Extract Supplemented With DMAP

[0269] This example illustrates the use of diluted CSF extractsupplemented with DMAP to achieve nuclear envelope formation and nuclearstructure in the absence of DNA synthesis. Xenopus erythrocyte nucleiwere isolated and pretreated using lysolecithin and trypsin as describedin Example 2 above. “Prepared CSF extract” was made using high speedcentrifugation and frozen by spotting the extract, made 7.5-10% (v/v)glycerol, as a 20 μl droplet onto a block of aluminum immersed in liquidnitrogen. Aliquots of the extract were thawed on ice and supplementedwith 10 mM creatine phosphate, 10 μg/ml creatine phosphokinase, 80 mMβ-glycerol-PO₄, and 0.1 mM CaCl₂. While still on ice the CSF extractreceived a small volume ({fraction (1/33)}^(rd)) of DMAP to a finalconcentration of 5 mM, and was then diluted with different amounts of EBbuffer as follows: mixture 1, 100%=extract only no EB; mixture 2, 75%=3volumes extract+1 volume EB; mixture 3, 50%=1 volume extract+1 volumeEB; and mixture 4, 25%=1 volume extract+3 volumes EB.

[0270] Each of these mixtures was warmed to 25° C. and incubated for 15minutes. Pretreated nuclei were then added in {fraction (1/10)} thevolume to a final concentration of 2000 nuclei/μl. Samples from eachincubate were taken immediately (0), 60, 90, 120 minutes later andfixed, examined, and photographed. Nuclei treatment with dilutedCSF-DMAP resulted in greater swelling than nuclei treated with undilutedCSF-DMAP. Nuclei treated with mixture 3 had a larger extent of nuclearswelling, nuclear envelope formation, and chromatin decondensation, thannuclei treated with the other mixtures. Additional experiments usingP³²-dCTP and biotinylated-dUTP demonstrated that no DNA synthesis tookplace during the process of nuclear swelling described above.

Example 9 Use of Diluted CSF Extract Supplemented With DMAP, MgCl₂ andEGTA

[0271] This example illustrates the effect of diluted CSF extractsupplemented with DMAP, MgCl₂ and EGTA on nuclear envelope formation,swelling, and chromatin structure. Nuclei were treated as described inExample 7 prior to the addition of CSF extract. The CSF extract(prepared as in Example 7), while still on ice was supplemented asfollows: 5 mM DMAP, 16 μM Biotinylated-dUTP and 16 μM MgCl₂. Thesupplemented extract was diluted with an equal volume of EB buffercontaining 5 mM potassium EGTA, pH 7. This mixture was warmed to 25° C.and incubated for 15 minutes. Pretreated nuclei were then added in{fraction (1/10)} the volume to a final concentration of 2000 nuclei/μl.Samples from each incubate were taken immediately 0, 15, 45, 60, and 90minutes later and were fixed, examined, and photographed.

[0272] The 50% CSF-DMAP, supplemented with. EGTA and MgCl₂, causedpretreated erythrocyte nuclei to rapidly swell and acquire a nuclearenvelope. No biotin incorporation into DNA was observed. Thus incontrast to Example 7, swelling took place in the absence of DNAsynthesis. In addition, the DNA observed in this example was morecompacted than the DNA observed in example 8. The difference betweenthis example and example 8 is likely due to the alteration of CSFextract cation concentration, and composition. For example, the EGTA maychelate the Ca²⁺ thereby lowering the Ca²⁺ while additional Mg²⁺ isadded to increase the Mg²⁺ concentration.

Example 10 Microchamber Microscope Slide

[0273] This example illustrates the use of the microchamber microscopeslide to analyze and activate nuclei. Xenopus erythrocyte nuclei wereisolated and pretreated as described above in Example 1. These nuclei,in NIB buffer, were allowed to settle onto the lower surface of severalmicrochamber microscope slides. A coverslip was placed over each sampleand sealed using oil along the sides. Nuclei were further pretreatedusing thawed “prepared CSF extract” made 80 mM in β-glycerol-PO₄ andsupplemented with 10 mM creatine phosphate and 10 μg/ml creatinephosphokinase. Ten microliters of CSF extract was allowed to flow intoeach well and the microchamber microscope slide was then subjected tothe following warm-then-cold treatment; 30 minutes on ice, 30 minutes at25° C., and 30 minutes on ice. After the warm-then-cold treatment, theCSF extract in each well was displaced by the addition of 20 μl freshly“prepared activating egg extract” containing biotinylated-dUTP. Themicrochamber microscope slides were then warmed to 25° C. At varyinglengths of time the incubations were stopped by rinsing the microchamberwith 75 μl of an appropriate buffer containing Texas red streptavidin(for detection of incorporated biotin), followed by staining withHoechst stain for detection of total DNA. The nuclei were photographedat a magnification of 60× using fluorescent optics.

[0274] Red blood cell nuclei before pretreatment were small and compact.The majority of nuclei were separated from one another indicating theywere not damaged or clumped during isolation.

[0275] Red blood cell nuclei at the end of further pretreatment in CSFextract were attached to the surface of the microchamber microscopeslide and remained small and highly compact.

[0276] Red blood cell nuclei 30 minutes after addition of activating eggextract swelled dramatically, and were attached to the surface of theslide. Texas red streptavidin staining of these nuclei demonstrated thelack of DNA replication.

[0277] Nuclei after 85 minutes of incubation in activating egg extractwere swollen. As seen by Texas red streptavidin staining, these nucleiwere surrounded with a nuclear envelope and initiated DNA replication.

[0278] After 150 minutes of incubation in fresh activating egg extractDNA replication was complete and the nuclei entered mitosis. As a resultof entering mitosis, the nuclear envelopes dissembled and the DNAcondensed into chromosome-like structures which remained attached to thesurface of the microchamber microscope slide.

[0279] These results demonstrate the utility of a microchambermicroscope slide in nuclei activation. Using the methods and productsdisclosed in the present invention nuclei were conveniently activated ona microchamber microscope slide.

Example 11 Activation of Nuclei from Fetal Red Blood Cells Isolated fromthe Umbilical Cord

[0280] The activation of human fetal red blood cells using activatingegg extract is described below. Human fetal red blood cells wereprepared from umbilical cord blood, pretreated with lysolecithin andtrypsin, and contacted with activating egg extract.

[0281] Human fetal red blood cells were isolated from umbilical cordblood and fractionated into a nucleated cell fraction and anon-nucleated cell fraction as described by Bianchi et al., Proc. Natl.Acad. Sci. USA 87:3279 (1990). A sample of neonatal umbilical cord bloodwas drawn into a vacuum tube containing anticoagulants, the blood wasdiluted 1:1 with Hank's balanced salt solution (HBSS) (Hanks andWallace, Proc. Exo. Biol. Med. 71:196 (1949)), layered over aFicoll/Hypaque column (Pharmacia) and spun at 1400 rpm for 40 min atroom temperature. The mononuclear cell layer was recovered and washedtwice by centrifugation in HBSS. The cells were then washed severaltimes in NIB buffer (250 mM sucrose, 25 mM NaCl, 10 mM Pipes, 1.5 MMMgCl₂₁ 0.5 mM spermidine, and 0.15 mM spermine, pH 7.0); the resultingcell pellet was suspended in NIB:Glycerol (7:3) and frozen in liquidnitrogen as 100 μl aliquots containing 6.3×10⁶ cells.

[0282] Frozen cells were thawed at room temperature and put on ice,washed twice with NIB, diluted to 4×10⁷ cells/ml in NIB at 23° C., andadded to an equal volume of NIB containing 80 μg/ml lysolecithin and 0.6μg/ml trypsin; lysolecithin and trypsin treatment was halted after 5minutes by adding soybean trypsin inhibitor to a concentration of 30μg/ml and bovine serum albumin to a final concentration of 0.4%.Isolated nuclei were added directly to thawed “prepared activating eggextract” to a concentration of 200 nuclei/μl, supplemented with 10 mMcreatine phosphate, 10 μg/ml creatine phosphokinase, 5 μg/ml nocodazole,0.3 mM cAMP, and 1 mM caffeine. One aliquot of this sample wassupplemented with P³²-dCTP at approximately 200 μCi/ml and used tomeasure DNA replication. A second aliquot was sampled periodically forfluorescent microscopic examination of nuclei after fixation andstaining with Hoechst dye (as described above).

[0283] Human red blood cell nuclei treated in the manner described aboveswelled significantly during the first 90 minutes and initiated DNAsynthesis. DNA synthesis continued for approximately 4.0 hours afterwhich nuclear chromatin condensed. However, the observed kinetics of DNAsynthesis indicated that complete genome replication was not achieved inthis experiment. The failure to achieve complete genome replication wasprobably due to the failure to further pretreat the isolated humannuclei in CSF extract and because activating egg extract contained arelatively high level of nocodazole, i.e., 5 μg/ml.

[0284] Despite the difficulties encountered, the formation of metaphasechromosomes demonstrates that the present invention can be used toactivate non-dividing human nuclei. The non-dividing human nucleiactivated analogously to Xenopus erythrocyte nuclei. Therefore, thevarious improvements described in the present invention, such as furtherpretreatment in CSF extract, a warm-then-cold regime and the addition of100 μM CaCl₂, which result in increasing the activation of non-dividingXenopus erythrocyte are applicable to activate non-dividing humannuclei.

Example 12 Activation of Nuclei of Fetal Red Blood Cells Isolated fromFetal Liver

[0285] This example illustrates the use of the products and methodsdescribed herein to determine preferred activation conditions andactivate blood cells isolated from fetal liver. Mononucleated cellsisolated from fetal liver, studied in this example, were predominatelyfetal blood cells as judged by their red color. The following steps werepreformed:

[0286] Step 1: Isolation of mononucleated human liver cells.Mononucleated human cells were isolated from human fetal liver by gentlytrimming the tissue and then homogenizing it between two glass slides.The cells were collected by suspension in phosphate buffered saline andthen transferred to a centrifuge tube. 2 ml of Ficoll was layered underthe cell suspension which was then centrifuged at 2000 rpm for 20minutes. The red mononuclear cells (upper layer) containingpredominantly erythroid blood cells were collected, diluted withphosphate buffered saline and centrifuged gently to pellet the cells.Cell pellets were resuspended and pelleted one more time in phosphatebuffered saline and then resuspended in RPM1 tissue culture medium.

[0287] The cells were frozen in liquid nitrogen. To prepare cells forfreezing in liquid nitrogen, the cells were pelleted by gentlecentrifugation, resuspended, and centrifuged again in Hank's balancedsalt buffer containing protease inhibitors (TPCK 0.1 mM, TLCK 0.1 mM,PMSF 0.05 mM, and leupeptin 5 μg/ml) at 4° C. The resulting supernatantwas clear indicating the absence of hemolysis. The pellet wasresuspended in 1-1.5 ml of NIB containing protease inhibitors (TPCK 0.1mM, TLCK 0.1 MM, PMSF 0.05 mM, and leupeptin 5 μg/ml), the volume wasbrought to 10 ml. The suspension was then spun at 1000 rpm for 10 minuteat 4° C., again no hemolysis was observed, and resuspended to finalvolume of 5 ml in NIB containing protease inhibitors (TPCK 0.1 mM, TLCK0.1 mM, PMSF 0.05 mM, leupeptin 5 μg/ml). The concentration of cells wasapproximately 3.55×10⁷/ml. The cells were then spun down, resuspended in1.775 ml 70% NIB-30% glycerol, and frozen as 50 μl aliquots in liquidnitrogen.

[0288] Step 2: Membrane permeabilization of nuclei.

[0289] The membrane of nuclei prepared as described in step 1 waspermeabilized using lysolecithin. Frozen cells were warmed quickly,diluted with NIB, and lysed by addition of lysolecithin at a finalconcentration of 40 μg/ml for 5 minutes at 25° C. At this point thenuclei are surrounded by a cytoskeletal matrix and do not expand ordivide if contacted with activating extract.

[0290] Step 3: Removal of cytoskeletal proteins surrounding the nucleusand nuclear matrix proteins within the nucleus.

[0291] The nuclei from step 2 were treated with trypsin using variableamounts of enzyme and treatment times. Increasing the length of trypsintreatment from 0-15 minutes, at 25° C., increased the extent of DNAsynthesis after standard CSF pretreatment and replication in activatingextract. Incubation times longer than 15 minutes resulted in decreasedreplication, probably due to nuclear damage and clumping. Optimaltrypsin pretreatment used 0.4 μg/ml trypsin for 15 minutes at 25° C.However, as would be appreciated by one skilled in the art, conditionsfor trypsin treatment may vary depending on how the cells are washed toremove protease inhibitors added during cell preparation and the trypsinincubation temperature. These results confirm that, human cell nuclei,like the Xenopus erythrocyte system, should be prepared for activationusing a controlled proteolytic step.

[0292] The lysolecithin-trypsin pretreatment was stopped by adding BSAto 0.4%+soybean trypsin inhibitor to a final concentration of 30 μg/mlfollowed by gentle centrifugation. The nuclear pellet was suspended andpelleted once more in 0.4% BSA and then in NIB alone.

[0293] Step 4: Further pretreatment with CSF extract.

[0294] Washed nuclei were further pretreated in CSF extract to enhanceactivation. Pretreatment as described in steps 2 and 3 was notsufficient to allow swelling and replication of nuclei usingfrozen/thawed activating extracts. This was attributed to the absence ofMPF activity in frozen/thawed activating extract. Indeed, pretreatmentwith CSF extract substantially increased responsiveness oflysolecithin/trypsin pretreated nuclei.

[0295] CSF extract further pretreatments were also preformed usingvarying lengths of time and temperature. Frozen/thawed CSF extract weresupplemented with 10 mM creatine phosphate, 10 μg/ml creatinephosphokinase, 80 mM β-glycerol-PO₄, and 0.1 mM CaCl₂. H1 kinase levelsin such extracts were high and stable for several hours.

[0296] An incubation for 60 minutes at 25° C. followed by 60 minutes at4° in CSF extract was found to give the highest amount of activation.Incubations at 25° C. for longer than 60 minutes resulted in lower DNAsynthesis, probably because individual nuclei break up into separatechromosomes. Nuclei in these experiments were at 2000/μl, but a broadrange of concentrations should be equally effective.

[0297] Additional experiments carried out using Xenopus erythrocytessuggest that the 60 minute cold incubation after the 60 minute warm stepincreases the rate of subsequent replication. Possibly, the cold stepdisassembles spindles containing microtubules that form around nucleiduring the warm step.

[0298] Thus, the CSF is preferably supplemented with 10mM creatinephosphate, 10 μg/ml creatine phosphokinase, 80mM β-glycerol-PO₄, and 0.1mM Ca²⁺, and a treatment regime involving incubation for 60 minutes at25° C. followed by 6 minutes at 4° C. is used in nuclei furtherpretreatment.

[0299] Step 5: Activation of erythroid cell nucleic Nuclear swelling,envelope formation, and replication were carried out on human fetalliver erythroid cell nuclei prepared using the optimal conditionsdescribed in steps 1-4 above. The nuclei were activated by diluting thefurther pretreated nuclei into nine volumes of prepared activatingextract (prepared from eggs activated for 30 minutes and supplementedwith 10 mM creatine phosphate, and 10 μg/ml creatine phosphokinase, plus16 μM biotinylated-dUTP or 0.2 μCi/μl αP³²-dCTP). The resulting nucleiconcentration was about 200/μl.

[0300] The following measurement were taken: DNA synthesis was monitoredusing extract containing P³²dCTP by gel electrophoresis; histone H1kinase was measured; and samples, labelled with biotinylated-dUTP weretaken for cytological analysis of DNA replication and nuclear envelopebreakdown.

[0301] DNA synthesis began after a lag of 30 minutes and continued until120 minutes. Little or no round-2 DNA synthesis occurred between 150-180minutes, probably because nuclear envelope breakdown had not takenplace. Second mitosis began between 180-210 minutes as seen by the risein H1 kinase activity, and was accompanied by fragmentation of the DNA.

[0302] Analysis of the size of the replicated DNA demonstrated that themolecules were initially very large, but at the time that DNA synthesisstopped (120 minutes) a substantial portion of the DNA was converted toa middle molecular weight (MMW) band of approximately 50 kilobases.Subsequent experiments have demonstrated that formation of this bandcoincides with onset of mitosis, even in the absence of a significanthistone H1 kinase peak, as is the case in this experiment. We believethat the MMW DNA band is an artifact generated by SDS-proteinase Kdisruption of the topoisomerase II-DNA complexes involved in chromosomecondensation. Daughter strand deconcatenation is a mandatory part ofchromosome condensation and of necessity requires Type-II topoisomerase(Topo II) breakage of the replicated chromosome at many sites. Thus webelieve that our in vitro conditions allow the normal G2-like period totake place following the S-phase. Chromosome condensation anddeconcatenation takes place during this period.

[0303] The process of DNA fragmentation process is distinct fromchromosome condensation and MMW band formation. Fragmentation appears tooccur when nuclei enter mitosis without having completed DNA synthesis,i.e., premature chromosome condensation.

[0304] Cytotological analysis demonstrated that little or no nuclearenvelope breakdown occurred in this experiment at 120-150 minutes, alsoin accord with the absence of a histone H1 kinase peak. Extensivenuclear swelling began about 30 minutes after incubation. Nuclearenvelopes formed between 30 and 60 minutes and biotin labelling of newDNA began by 60 minutes. The intensity of biotin labelling increasedduring the S-phase, in keeping with p³² labelling. Maximum swelling withchromatin dispersion was reached at about 90 minutes, while somecondensation of chromatin took place at about 90-120 minutes although asmall amount of DNA synthesis was still on going. In the period 120-150minutes there was marked chromatin condensation suggesting the onset ofmitotic prophase, but nuclear envelope breakdown did not take place.

[0305] The 90 minute sample was further analyzed to determine the extentof sample homogeneity. The first 36 nuclei detected with Hoechst stainwere photographed. In replicated nuclei Texas red staining of biotinbleeds through into the blue Hoechst channel turning the nuclei purple.About 40% of the nuclei failed to swell and failed to replicate. Almostall remaining nuclei swelled and replicated to the full extent. Therewere very few partially replicated nuclei. The inability of some of thenuclei to replicate was attributed to nuclear damage since the responseof carefully prepared frog erythrocyte nuclei is much more homogeneous.

[0306] Experiments were also carried out to determine whether nuclearswelling was dependent on DNA synthesis, by activating nucleic in thepresence of aphidicolin (added at T=0). Biotin labelling confirmed thatno replication took place in the aphidicolin treated nuclei. The resultsdemonstrated that even in the absence of replication many nuclei swelledsignificantly.

[0307] Step 6: Formation of mitotic chromosomes.

[0308] Swelled and replicated nuclei were treated with Cyclin-

90 or CSF extract. Cyclin-

90 was added to activating extract to obtain prophase mitoticchromosomes, while CSF extract was added to obtain metaphase chromosomesand nuclear envelope breakdown.

[0309] Addition of {fraction (1/20)}^(th) volume of cyclin-

90 at T=100 (minutes) caused a slight but real improvement in theclarity of prophase chromosomes observed in T=120 nuclei and thereafterseemed to increase the extent of chromosome condensation. Nuclearenvelope breakdown was observed at T=240 and separate, chromosome-likewere released.

[0310] Addition of ½ volume of CSF extract at T=100 caused a rapidextensive condensation of DNA and disappearance of the nuclear envelope.This state of condensation remained stable until T=240.

Example 13 Activation of Human Sperm Nuclei Using a PPT Pretreatment

[0311] This example illustrates the use of apermeabilization-protease-thiol reducing agent (PPT) pretreatment toenhance activation of human sperm nuclei. Fresh semen from a healthymale donor was obtained, diluted in an equal volume of yolk test buffer(Jasjey, D. G. and Cohen, M. R. Fertility Sterility 35:205-212, 1981)and frozen in liquid nitrogen in approximately 1 ml aliquots containingapproximately 1×10⁸ sperm of which approximately 68% were motile.

[0312] On the day of the experiment, sperm samples were thawed at roomtemperature and washed twice in ice cold NIB buffer by centrifugation.Sperm were then permeabilized by incubation in 100 μg/ml lysolecithinfor 5 minutes at 25° C. and then treated with 100 μg/ml trypsin (e.g., aprotease) for 10 minutes at the same temperature. Thelysolecithin/trypsin treatment was stopped by the addition of soybeantrypsin inhibitor to 30 μg/ml and dialyzed/lyophilized bovine serumalbumin to 0.4% and then washed by centrifugation. The sperm were thenincubated in a solution of 5 mM dithiothreitol (DTT) (e.g., a thiolreducing agent) in 5 mM in NIB for 60 minutes on ice and thenpost-treated with 1 mM N-ethylmaleimide in NIB for 10 minutes at 25° C.A final wash was carried out in NIB and the nuclei were resuspended at40,000/μl. Two additional aliquots were prepared as described aboveexcept that in one case the DTT was omitted from the 60 minuteincubation in NIB, and in the other case the trypsin was omitted duringthe 10 minute incubation following lysolecithin treatment.

[0313] Each of the above three samples were then added at a finalconcentration of 4000/μl to a frozen/thawed preparation of high speed“prepared CSF extract” that had been supplemented with 10 mM creatinephosphate, 10 μg/ml creatine phosphokinase, 80 mM β-glycerophosphate,and 0.1 mM CaCl₂. Nuclei were incubated for 90 minutes at 25° C. andthen for 60 minutes at 4° C.

[0314] Each of the three CSF extract treated samples were then dilutedwith 9 volumes of a frozen/thawed 25 minute activated egg extractsupplemented with 10 mM creatine phosphate, and 10 μg/ml creatinephosphokinase, plus 16 μM biotinylated-dUTP and 16 μM MgCl₂, or plus 0.2μCi/μl αP³²-dCTP. Each sample was incubated at 25° C. and sampledperiodically by either fixed staining for biotin incorporation into DNAand photographed, or fractionated on agarose gel and counted forincorporation of label nucleotides into DNA.

[0315] Cytology demonstrated that each of the three treatment regimesresulted in swelling of the sperm nuclear DNA, but only the combinedlysolecithin-trypsin-DTT pretreatment procedure resulted in new nuclearenvelope formation, extensive spherical swelling of the nucleus, and newDNA synthesis. DNA synthesis as determined by P³²-dCTP incorporationalso demonstrated that only the combined lysolecithin-trypsin-DTTpretreatment procedure resulted in significant replication. The combinedlysolecithin-trypsin-DTT pretreatment procedure brought more than a 5fold increase in DNA synthesis than pretreatments with lysolecithin andtrypsin or lysolecithin and DTT.

[0316] Additional cytological analysis and in situ hybridization wascarried out using two nucleic acid probes; one to a reiterated sequenceon the X-chromosome and one to a single copy sequence on chromosome 18.The lysolecithin-trypsin-DTT pretreated and activated sperm nuclei wererecovered after 120 minutes of incubation in activating extract in thepresence of biotinylated-dUTP. Two nuclei were stained for total DNA(Hoechst=blue), the X-chromosome (using a digoxygenin-labelledprobe-green), newly synthesized biotinylated DNA (Texas-redstreptavidin=red), and chromosome 18 (using a biotinylatedprobe+Texas-red streptavidin=red dots). The intensity of the Hoechststaining and the biotinylated-dUTP staining in the first nucleus wasless than that in the second nucleus. This demonstrates that firstnucleus had only begun in vitro replication while the second nucleus hadreplicated more completely. In addition, the first nucleus containedonly one copy of the X-chromosome, indicating that the probed region ofthe chromosome had not yet replicated, while the second nucleuscontained two copies of the X-chromosome, indicating that it hadreplicated by this time. In addition, two copies of chromosome 18 weredetected in the first nucleus, suggesting that this probe detects anearly replicating sequence. Detection of chromosome 18 was obscured inthe in the second nucleus by the higher level of incorporatedbiotinylated-dUTP.

[0317] These results are consistent with the notion that both thiolreduction and protease induced changes in the sperm cytoskeleton andprotamines are required for displacement of protamines and theirreplacement by chromatin forming histones in the egg extracts. Chromatinassembly is important in the formation of the surrounding nuclearenvelope and completion of the envelope is important for initiation ofDNA synthesis. These results also demonstrated that human sperm cellnuclei which have been activated and replicated in vitro can be used forgenetic analysis.

[0318] Additional experiments demonstrated that the optimized protocoldescribed above also resulted in formation of mitotic chromosomes fromhuman sperm nuclei.

[0319] Other embodiments are within the following claims.

1. A method for activating a nucleus from a human fetal cell comprisingthe steps of: a) separating said nucleus from its surroundingcytoskeleton to form a pretreated nucleus, and b) contacting saidpretreated nucleus with an activating egg extract to activate saidpretreated nucleus.
 2. The method of claim 1, wherein said fetal cell isselected from the group consisting of keratinocyte, trophoblast,erythrocyte and leukocyte.
 3. The method of claim 2, wherein saidleukocyte is selected from the group consisting of neutrophil,basophile, eosinophil, and granulocyte.
 4. The method of claim 1,wherein said separating is carried out using a protease and a non-ionicdetergent.
 5. The method of claim 4, wherein said protease is trypsinand said detergent is lysolecithin.
 6. The method of claim 1, furthercomprising contacting said pretreated nucleus with CSF extract.
 7. Themethod of claim 6, wherein said nucleus is activated under conditionswere the nucleus and its chromosomes do not divide.
 8. The method ofclaim 7, wherein said conditions comprise adding nocodazole to saidactivating egg extract or said CSF extract.
 9. The method of claim 8,wherein said nocodazole is in amount less than 5 μg/ml.
 10. The methodof claim 1, wherein said nucleus is activated under conditions notsuitable for nucleic acid: synthesis.
 11. A method of causing anon-dividing human nucleus to activate, using activating egg extract andCSF extract prepared from hardened eggs comprising: a) incubating saidnon-dividing human nucleus with said CSF extract prepared from hardenedeggs to form a pretreated nucleus, and b) contacting said pretreatednucleus with said activating egg extract wherein said activating eggextract is prepared from synchronously activated hardened eggs.
 12. Themethod of claim 11, wherein said CSF extract is frozen and thawed beforeuse.
 13. The method of claim 11, wherein said activating egg extract isfrozen and thawed before use.
 14. The method of claim 11, wherein saidincubating is performed using a warm-then-cold regime comprisingincubating at about 25° C. for at least 30 minutes followed byincubation at about 4° C. for at least 30 minutes.
 15. The method ofclaim 11, wherein said incubating is performed using a warm regime atabout 25° C. for at least 30 minutes.
 16. The method of claim 11,wherein Ca²⁺ is provided in said incubating step.
 17. The method ofclaim 16, wherein said Ca²⁺ is provided in an amount greater than 100μM.
 18. A method for preparing an activating egg extract, comprising thesteps of hardening a plurality of eggs, simultaneously inducing saideggs; and preparing an activating egg extract from said eggs whereinsaid eggs are induced for a length of time such that they have at least70% of maximal activation DNA synthesis activity.
 19. The method ofclaim 18, wherein said activating egg extract is prepared from aeukaryotic cell.
 20. The method of claim 19 wherein said eukaryotic cellis an amphibian, yeast, human, echinoderm, mollusc, fish, or chickencell.
 21. The method of claim 20, wherein said eukaryotic cell is aXenopus cell.
 22. The method of claim 21, wherein said eggs are obtainedfrom Xenopus and said length of time is greater than 10 minutes.
 23. Themethod of claim 22, wherein said length of time is between 25 and 30minutes.
 24. A method of causing a non-dividing cell nucleus to swellcomprising the steps of: a) separating said nucleus from its surroundingcytoskeleton to form a pretreated nucleus; and b) contacting saidpretreated nucleus with a CSF extract supplemented with an aqueoussolution or a protein kinase inhibitor.
 25. The method of claim 24,wherein said nucleus is a human nucleus.
 26. The method of claim 25,wherein said CSF extract is a partially purified CSF extractsupplemented with an aqueous solution and a protein kinase inhibitor.27. The method of claim 26, wherein said aqueous solution is anappropriate buffer.
 28. The method of claim 27, wherein said proteinkinase inhibitor is either 6-dimethylaminopurine or staurosporine. 29.The method of claim 28, wherein said appropriate buffer is provided inan amount to dilute said CSF extract by 25% to 75%.
 30. The method ofclaim 27, further comprising the step of adding an agent to furtherswell or decondense said nuclei after said step (b).
 31. A method ofcausing chromosome formation in a non-dividing cell nucleus comprisingthe steps of: a) separating said nucleus from its surroundingcytoskeleton to form a pretreated nucleus; and b) contacting saidpretreated nucleus with a CSF extract supplemented with a cyclin. 32.The method of claim 31, wherein said cyclin is cyclin-


90. 33. A method for activating or studying a mammalian sperm cellnucleus comprising the steps of: a) pretreating said sperm cell nucleusto form a pretreated sperm nucleus wherein said pretreating comprises(i) separating said nucleus from its surrounding cytoskeleton bypermeabilizing said cell nuclear membrane and incubating in the presenceof a protease and (ii) incubating in the presence of a thiol reducingagent; and b) activating said pretreated sperm cell.
 34. The method ofclaim 33 wherein said sperm cell nucleus is a human sperm cell nucleus.35. The method of claim 34, wherein said activating is carried out bycontact with a CSF extract, wherein said CSF extract is supplemented toinduce nuclear swelling.
 36. The method of claim 34, wherein saidactivating is carried out by contact with a CSF extract, wherein saidCSF extract is supplemented to induce chromosome formation.
 37. Themethod of claim 34, wherein said activating is carried out using anactivating extract.
 38. The method of claim 33, wherein said sperm cellnucleus is from a transgenic animal.
 39. The method of clam 34, whereinsaid human sperm cell nucleus is obtained from either a person infectedwith a virus or a person not infected with said virus.
 40. The method ofclaim 39 wherein, said virus is HIV.
 41. The method of claim 34, furthercomprises the step of analyzing said nucleus by in situ hybridization.42. The method of claim 34, wherein said step (a) (i) is carried outusing a detergent and trypsin.
 43. The method of claim 34, wherein saidpretreating step further comprises a thiol blocking agent.
 44. Themethod of claim 34, wherein a CSF extract further pretreatment step iscarried out between said step (a) and said step (b), comprisingincubating the pretreated nuclei in CSF extract.
 45. An activation assayfor studying male fertility comprising: a) pretreating a sperm nucleusto separate cytoskeletal protein from nucleic acid, b) activating saidsperm nucleus, c) measuring activation of said nucleus activated in step(b).
 46. The activation assay of claim 45, wherein said pretreatingcomprises using a detergent, a protease, and a thiol reducing agent. 47.The activation assay of claim 46, wherein said sperm nucleus is a humansperm nucleus.
 48. The activation assay of claim 47, wherein saidactivating is carried out using a CSF extract supplemented with anaqueous solution, a protein kinase inhibitor, or a cyclin.
 49. Themethod of claim 47, wherein said activating is carried out using anactivating egg extract.
 50. The method of claim 49, further comprising aCSF further pretreatment prior to said activating.
 51. A viralintegration assay comprising the steps of: a) pretreating a cell nucleusto separate the nucleus from its surrounding cytoskeleton to form apretreated nucleus, b) activating said nucleus and incubating with aviral integration complex containing viral nucleic acid, wherein saidintegration complex is added before or after said incubating; and c)measuring integration of viral nucleic acid into nucleic acid of saidcell nucleus.
 52. The assay of claim 51, wherein said viral nucleic acidis from HIV.
 53. The assay of claim 52, further comprising a step,between said step b) and said step c), of adding an agent which inhibitsintegration of said HIV.
 54. A viral integration assay comprising thesteps of: a) constructing a pseudonucleus from a defined DNA template,b) activating said nucleus and incubating with a viral integrationcomplex containing viral nucleic acid, wherein said integration complexis added before or after said incubating; and c) measuring integrationof viral nucleic acid into nucleic acid of said cell nucleus.
 55. Aproduct for preparing a non-dividing nucleus to activate upon subsequenttreatment with activating egg extract, comprising a CSF extract preparedusing an eukaryotic cell, wherein said CSF extract is supplemented withCa²⁺.
 56. The product according to claim 55, wherein said Ca²⁺ isprovided in an amount greater than 100 μM.
 57. A product for causingnuclear swelling comprising CSF extract supplemented with a proteinkinase inhibitor and/or an aqueous solution.
 58. The product of claim57, wherein said CSF extract is a partially purified extract.
 59. Theproduct of claim 58, wherein said aqueous solution is an appropriatebuffer.
 60. The product according to claim 59, wherein said supplementis said protein kinase inhibitor and said appropriate buffer.
 61. Theproduct according to claim 60, wherein said protease inhibitor is either6-dimethylaminopurine or staurosporine.
 62. A product for causingchromosome formation in a cell nucleus comprising a CSF extractsupplemented with a cyclin.
 63. The product according to claim 62,wherein said cyclin is cyclin-


90. 64. A product for activating a non-dividing nucleus comprising anactivating egg extract having at least 70% optimal activation activity.65. The product of claim 64, wherein said activating egg extract isprepared from one or more eukaryotic egg.
 66. The product of claim 64,wherein said activating egg extract is prepared from a plurality ofXenopus eggs synchronously induced for more than 10 minutes.
 67. Theproduct of claim 66, wherein said Xenopus eggs are synchronously inducedfor 25 to 30 minutes.
 68. The product of claim 66, wherein saidactivating egg extract is supplemented with cAMP.
 69. The product ofclaim 66, wherein said activating egg extract is supplemented with aphosphodiesterase inhibitor.
 70. A kit for activating a non-dividingnucleus comprising a first product comprising frozen activating eggextract having at least 70% optimal activation activity and a secondproduct comprising a frozen CSF extract.
 71. The kit according to claim70, wherein said second product is supplemented with Ca²⁺.
 72. The kitaccording to claim 70, wherein said first product and said secondproduct are prepared from hardened eggs.
 73. The kit according to claim70, further comprising a microchamber microscope slide.
 74. A microscopeslide comprising; a) an upper surface, b) a water repellent-means havinga defined thickness located upon said upper surface to define amicrochamber connected by a channel to at least one well on said uppersurface, and c) said microchamber shaped to enhance flushing of saidmicrochamber.
 75. The microscope slide of claim 74, wherein saidmicrochamber is in a teardrop-shape or a pear-shape.
 76. The microscopeslide of claim 74, wherein two wells are provided at opposite ends ofsaid microchamber and each of said wells are connected to saidmicrochamber by a channel.
 77. The microscope slide of claim 74, whereinsaid microchamber has a defined volume between 5 and 50 μl.
 78. Themicroscope slide of claim 74, wherein said microchamber has definedvolume between 10 and 20 μl.
 79. The microscope slide of claim 74,wherein said water repellent means is a tape or a coating on said uppersurface of said slide.
 80. The microscope slide of claim 74, whereinsaid water repellent means is a TEFLON® coating.
 81. The microscopeslide of claim 74, wherein said water repellent means is a plasticcontaining tape.
 82. The microscope slide of claim 74, wherein saidupper surface is treated to enhance cell growth compared to an untreatedslide.
 83. The microscope slide of claim 74, wherein said microscopeslide is sterile.
 84. The microscope slide of claim 74, wherein saidmicrochamber or said at least one well contains an antibody to a humanfetal cell.
 85. A method for analysis of growth or manipulation of acell or cell component comprising providing a microscope slide of claim74 and placing said cell or said cell component in said microchamber.86. The method of claim 74, wherein a fluid is introduced into one saidwell and is allowed to enter said microchamber and then is removed fromsaid microchamber.